C-type lectin polypeptide, polynucleotide and methods of making and use thereof

ABSTRACT

Provided herein are polypeptide and polynucleotide sequences for a molecule having homology to the C-type lectin family of polypeptides. Also provided are methods of making and using the polypeptide and polynucleotides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/079,183, filed Mar. 25, 2008, now U.S. Pat. No. 7,695,937, whichis a divisional application of U.S. application Ser. No. 10/499,184,filed Jun. 17, 2004, now U.S. Pat. No. 7,396,662, which is a nationalstage filing under 35 U.S.C. §371 of international applicationPCT/US02/41158, filed Dec. 19, 2002, which claims the benefit of U.S.provisional application Ser. No. 60/342,001, filed Dec. 19, 2001, theentire disclosure of which is relied upon and incorporated by referenceherein.

FIELD OF THE INVENTION

The invention is directed to novel substantially purified polypeptidesof the C-type lectin family of polypeptides and soluble fragmentsthereof, polynucleotides encoding such polypeptides and fragments,processes for production of recombinant forms of such polypeptides andfragments, antibodies generated against these polypeptides or fragments,and assays and methods employing these polypeptides, antibodies, andpolynucleotides.

BACKGROUND

Calcium-dependent lectins (C-type lectins) are expressed in a largenumber of cell types including macrophages, B- and T-lymphocytes, mastcells, and natural killer (NK) cells. Macrophage lectin proteins performa variety of functions in the recognition and destruction of foreigncells and pathogens. Gram positive and Gram negative bacteria have beenshown to interact with C-type lectins (Athamna et al., Infect Immun59:1673, 1991; Shimaoka et al., J. Immunol. 166(8):5108, 2001). A humanmacrophage C-type lectin has been found to recognize Tn Ag, a well-knownhuman carcinoma-associated epitope (Suzuki et al., J Immunol 156:128,1996). Furthermore, the recombinant cytosolic carbohydrate bindingdomain of the mouse macrophage C-type lectin also served as an inhibitorof cytotoxic activity, indicating that the lectin was a direct mediatorof the macrophage tumoricidal response (Imai et al., J Immunol Methods171:23, 1994). Unique macrophage lectins may specifically interact withsurface antigens expressed by certain abnormal or diseased cells. Thelectins may direct the macrophages to abnormal or diseased cells.

Conserved features of C-type lectins include an extracellularcarbohydrate recognition domain (Spiess, M Biochem 29:10009, 1990).Receptor proteins of the C-type lectin superfamily do not generallyshare significant sequence homology beyond that of the carbohydraterecognition domain. C-type lectins are typically Type II membraneproteins. Type II membrane proteins include an extracellular C-terminusthat has the carbohydrate binding domain, an amino terminal cytoplasmicdomain, and a membrane-spanning domain of approximately 20 residues.Several prolines generally precede the cytoplasmic domain of thetransmembrane domain. The prolines are thought to prevent stericinterference of the amino-terminal domain with the transmembrane domainduring membrane insertion. The N-terminal cytosolic domains of theC-type lectins are very diverse in both length and sequence.Phosphorylation of tyrosine in the cytosolic domain of theasialoglycoprotein receptor, a C-type lectin, has been demonstrated(Fallon, J Biol Chem 265:3401, 1990). The extracellular carbohydratebinding domains can be separated from membrane-bound C-type lectinmolecules by protease treatment. These isolated, soluble domains retainstructural integrity and carbohydrate binding activity, owing in part tothe three-intrachain disulfide bonds present in the binding domains ofthis class of lectin.

C-type lectins play a role in the recognition and destruction ofdiseased and non-self cells. The selective modulation of the expressionand specificity of novel C-type lectins may allow the successfulmanagement of diseases related to macrophage function, such as graftrejection or pathogen colonization, or the exploitation of the naturalcytolytic capabilities of macrophages, such as specific targeting totumors or infected host cells.

A number of groups have identified several new C-type lectins unique tomacrophages and DC, such as the murine macrophage-restricted C-typelectin (mMCL) (Balch, S., et al., J. Biol. Chem. 273:18656-64, 1998);Langerin, the Langerhans cell-specific C-type lectin (Valladeau, J.,Immunity 12:71-81, 2000); Mincle, a macrophage-inducible C-type lectinthat is a transcriptional target of NF-IL6 in murine peritonealmacrophages (Matsumoto, M., et al., J. Immunol. 163:5039-48, 1999);DCIR, the human dendritic cell immumoreceptor, a type II glycoproteinwith homology to the macrophage lectin and hepatic asialoglycoproteinreceptors (Bates, E., et al., J. Immunol. 163:1973-83, 1999 and U.S.Pat. No. 6,277,959, both of which are incorporated herein by reference);and, murine Dectin-1 and Dectin-2 (DC-associated C-type lectins;Ariizumi, K., et al., J. Biol. Chem., 275:20157-167, 2000 and Ariizumi,K., et al., J. Biol. Chem., 275:11957-963, 2000, respectively), whichare thought to be involved in delivering T-cell costimulatory signals.

The present invention identifies additional, novel C-type lectinpolypeptides.

SUMMARY OF THE INVENTION

The present invention relates to a novel human C-type lectin, referredto herein as a C-type lectin-like (CtLL) polypeptide or polynucleotide.

The invention provides a substantially purified polypeptide comprising asequence that is at least 80% identical to a sequence as set forth inSEQ ID NO: 2, 4, 6, 8, 10, 12, 14, and/or 16 and having oxidized lipidand/or carbohydrate binding activity; a polypeptide comprising a solublefragment of any one of these having carbohydrate binding activity; apolypeptide comprising a soluble fragment of any one of these havingoxidized lipid binding activity; and a polypeptide comprising a fragmentof any of one these having a lectin domain amino acid sequence.

The invention further provides a substantially purified solublepolypeptide comprising a sequence that is at least 80% identical to asequence selected from the group consisting of: from about residue 1 toabout 41 of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, or 16; from about residue66 to about 136 of SEQ ID NO:2, 8, 14, or 16; from about residue x₁ toabout x₂ of SEQ ID NO:4, wherein x₁ is a residue between and includingresidues 66 and 143 and x₂ is a residue between and including residue226 and 233; from about residue x₁ to about x₂ of SEQ ID NO:6, whereinx₁ is a residue between and including residues 66 and 143 and x₂ is aresidue between and including residue 227 and 232; from about residue x₁to about 247 of SEQ ID NO:10, wherein x₁ is a residue between andincluding about residues 66 and 142; and from about residue x₁ to about276 of SEQ ID NO:12, wherein x₁ is a residue between and includingresidues 66 and 142, wherein the soluble fragment comprises oxidizedlipid and/or carbohydrate binding activity.

The invention also provides a fusion polypeptide comprising apolypeptide of the invention operably linked to a leucine zipperpolypeptide, an Fc polypeptide, or a peptide linker.

The invention provides polynucleotides encoding the polypeptides andfusion polypeptides of the invention. In one embodiment, thepolynucleotide is selected from the group consisting of: apolynucleotide comprising a sequence that is at least 80% identical to asequence selected from the group consisting of: SEQ ID Nos:1, 3, 5, 7,9, 11, 13, and 15; a polynucleotide comprising a sequence that is atleast 80% identical to a sequence selected from the group consisting of:from about nucleotide 1 to about nucleotide 123 of SEQ ID NO:1, 3, or13; from about nucleotide 196 to about nucleotide 408 of SEQ ID NO:1 or13; from about nucleotide 478 to about nucleotide 489 of SEQ ID NO:1;from about nucleotide x₃ to about nucleotide x₄ of SEQ ID NO:3, whereinx₃ is a nucleotide between about 196 to about nucleotide 427 and x₄ is anucleotide between about 678 to about nucleotide 699; from about 427 toabout nucleotide 678 of SEQ ID NO:3; from about nucleotide 37 to aboutnucleotide 159 of SEQ ID NO:5; from about nucleotide x₃ to aboutnucleotide x₄ of SEQ ID NO:5, wherein x₃ is a nucleotide between about232 to about nucleotide 463 and x₄ is a nucleotide between about 717 toabout nucleotide 732; from about nucleotide 463 to about nucleotide 717of SEQ ID NO:5; from about nucleotide 49 to about nucleotide 171 of SEQID NO:7 or 11; from about nucleotide 244 to about nucleotide 456 of SEQID NO:7; from about nucleotide 526 to about nucleotide 576 of SEQ IDNO:7; from about nucleotide 101 to about nucleotide 223 of SEQ ID NO:9;from about nucleotide x₃ to about nucleotide 841 of SEQ ID NO:9, whereinx₃ is a nucleotide between about 296 to about nucleotide 524; from aboutnucleotide 524 to about nucleotide 841 of SEQ ID NO:9; from aboutnucleotide x₃ to about nucleotide 876 of SEQ ID NO:11, wherein x₃ is anucleotide between about 244 to about nucleotide 475; from aboutnucleotide 475 to about nucleotide 876 of SEQ ID NO:11; from aboutnucleotide 478 to about nucleotide 534 of SEQ ID NO:13; from aboutnucleotide 164 to about nucleotide 286 of SEQ ID NO:15; from aboutnucleotide 359 to about nucleotide 571 of SEQ ID NO:15; and from aboutnucleotide 641 to about nucleotide 690 of SEQ ID NO:15; a polynucleotidethat hybridizes under moderately stringent conditions to apolynucleotide comprising the sequence above; a nucleotide sequencecomplementary to the nucleic acid sequence of above; and any ofnucleotide sequences of above wherein T can also be U.

The invention further provides an expression vector comprising apolynucleotide of the invention as well as recombinant host cellsgenetically engineered to contain a polynucleotide or expression vectorof the invention.

The invention provides a method for producing a polypeptide of theinvention, comprising culturing the host cell containing apolynucleotide of the invention under conditions promoting expression ofthe polypeptide and purifying the polypeptide.

Antibodies that bind to a polypeptide of the invention are alsoprovided. In one embodiment, the antibody specification binds to apolypeptide having a sequence as set forth in SEQ ID NO:2, 4, 6, 8, 10,12, 14, and/or 16. The antibody can be monoclonal, polyclonal, human,humanized, and the like. In one embodiment the antibody inhibits theactivity of a polypeptide of the invention.

The invention further provides a method for identifying an agent thatmodulates an activity of a polypeptide of the invention. The methodincludes contacting an agent and a polypeptide of the invention underconditions such that the agent and polypeptide interact; and determiningthe activity of the polypeptide in the presence of the agent compared toa control, wherein a change in activity is indicative of an agent thatmodulates the polypeptide's activity. The agent can be an antibody, asmall molecule, a peptide, and/or a peptidomimetic.

Also provided by the invention is a method of inhibiting the formationof atherosclerotic plaques in a mammal in need of such treatment,comprising administering to the mammal an inhibition-effective amount ofa solCtLL polypeptide of the invention.

A method for inhibiting infection in a subject is also provided by theinvention. The method includes contacting the subject with a solCtLLpolypeptide of the invention.

The invention also provides a method for modulating endothelial cellmigration, comprising contacting an endothelial cell with a polypeptideof the invention. The contacting can be in vitro, ex vivo, or in vivo.

The invention further provides a method of modulating immune cellactivity comprising contacting a cell or mammal that expresses apolypeptide of the invention with an effective amount of solCtLLpolypeptide. In one embodiment, the mammal is afflicted with a conditionselected from the group consisting of ocular disorders; malignant andmetastatic conditions; inflammatory diseases; osteoporosis, acceleratedbone resorption disorders; restenosis; inappropriate plateletactivation, recruitment, or aggregation; thrombosis; atherosclerosis;and a condition requiring tissue repair or wound healing. In anotherembodiment, the solCtLL polypeptide is in the form of a multimer.

Also provided by the invention is a method of stimulating an immuneresponse in a subject. The method includes administering an agent to thesubject wherein the agent blocks the interaction of a CtLL polypeptidewith its ligand. In one embodiment, the agent is an antibody.

The invention also provides a method of inhibiting an immune response ina subject in need of such treatment comprising administering an agent tothe subject wherein the agent simulates CtLL activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of the CtLL polypeptides of the invention, SEQID NO: 8, 2, 16, 14, 10, 6, and 4 (top to bottom) and indicates thepolypeptides' putative domains.

FIG. 2 A-C shows a Pile-Up of molecules having homology to a CtLLpolypeptide SEQ ID NO: 12 of the invention.

DETAILED DESCRIPTION OF THE INVENTION

C-type lectins are a family of glycoproteins that exhibit amino acidsequence similarities in their carbohydrate recognition domains (CRD)and that bind to selected carbohydrates in a Ca²⁺-dependent manner.C-type lectins have been subdivided into four categories (Vasta et al.,Ann N Y Acad Sci., 712:55-73, 1994; Spiess, Biochemistry,29:10009-10018, 1990). The first group comprises type IImembrane-integrated proteins, such as asialoglycoprotein receptors,macrophage galactose and N-acetyl glucosamine (GlcNac)-specific lectin,and CD23 (FceRII). Many members in this group exhibit specificity forgalactose/fucose, galactosarnine/GalNac or GlcNac residues. The secondgroup includes cartilage and fibroblast proteoglycan core proteins. Thethird group includes the so-called “collectins” such as serummannose-binding proteins, pulmonary surfactant protein SP-A, andconglutinin. The fourth group includes certain adhesion molecules, whichare known as LEC-CAMs (e.g., Mel-14, GMP-140, and ELAM-1).

C-type lectins are known to function as agglutinins, opsonins,complement activators, and cell-associated recognition molecules (Vastaet al., Ann N Y Acad Sci., 712:55-73, 1994; Spiess, Biochemistry,29:10009-10018, 1990; Kery, Int J Biochem., 23(7-8):631-40, 1991). Forinstance, macrophage mannose receptors serve a scavenger function(Shepherd et al., Am J Respir Cell Mol Biol., 2(4):335-40, 1990), aswell as mediating the uptake of pathogenic organisms, includingPneumocystis carinii (Ezekowitz et al., Nature, 351(6322):155-8, 1991)and Candida albicans (Ezekowitz et al., J Exp Med., 172(6):1785-94,1990). Serum mannose-binding protein mimics Clq in its capacity toactivate complement through the classical pathway. Importantly, geneticmutations in this lectin predispose for severe recurrent infections,diarrhea, and failure to thrive (Reid et al., Springer SeminImmunopathol., 15(4):307-26, 1994). Thus, C-type lectins exhibit diversefunctions with biological significance.

Many diseases have been identified that relate to abnormalities ofmacrophage function, especially adherence, chemotaxis, and microbicidalactivity. Some of these abnormalities may be due to defects in therecognition of foreign particles, diseased tissues or host tissues vialectin receptor molecules expressed by, for example, macrophages. Forexample, macrophage cell-surface lectins may be linked to the chronicrejection of cardiac allografts in arteriosclerosis, by serving as apossible mediator of macrophage infiltration (Russel et al., J ClinInvest 94: 722-730, 1994). Pathogenic mycobacteria, including M.tuberculosis, colonize activated macrophages. The attachment of suchpathogens to macrophages is the preliminary step in pathogenesis. Thecolonization has been shown to occur via mannose-specific lectinreceptors expressed on the macrophages (Goswami et al., FEBS Lett355:183-186, 1994).

Based upon its homology to the C-type lectin protein family, the CtLLpolynucleotides and polypeptides disclosed herein may be useful in thetreatment or prevention of disorders such as graft rejection, autoimmunedisease, bacterial and parasitic infections, cell proliferativedisorders (e.g., cancer), and cardiac and vascular diseases anddisorders (e.g., myocardial infarctions and stroke associated withatherosclerosis) to name a few. Other diseases and disorders areprovided herein and are known to those of skill in the art.

The CtLL polypeptides of the invention lack residues important forbinding to carbohydrate ligands. The CtLL polypeptides have greatersequence homology to a related member of the C-type lectin family, OLR1and OLR1-related C-type lectin molecules (OLR1 was previously calledLOX-1). OLR1 is an endothelial receptor for oxidized low-densitylipoprotein that plays essential roles in atherogenesis and has a genestructure that resembles that of the natural killer cell receptors. TheCtLL polynucleotide sequence lies closes in the genome to CLEC2 (afamily member expressed in liver) and between CLEC2 and CLEC1 (a familymember expressed in dendritic cells and endothelial cells) in asub-cluster of molecules expressed in macrophages, DCs and endothelialcells including ORL1 which is expressed in macrophages and induced inendothelial cells and vascular smooth muscle cells (see also U.S. Pat.No. 6,277,959, the disclosure of which is incorporated herein byreference). The CtLL polynucleotide lies approximately 12 kB from CLEC2and about 150 kB from CLEC1 (although there is evidence there is anintervening gene in this area). The CtLL polypeptides of the presentinvention have not been detected on DC, however, they have been detectedon monocytes.

Based upon CtLL homology to the C-type lectins and OLR1, the CtLLpolypeptides of the invention are predicted to have activitiesassociated with oxidized low-density lipoprotein (oxLDL) binding and/orcarbohydrate binding. Oxidized lipids, in particular oxLDL, have beenshown to play a crucial role in atherosclerotic plaque formation and area major immunogenic target believed to be a factor in “hot” ordestabilized plaques that are a risk factor for thrombolytic eventsincluding lethal myocardial infarction and stroke. The progression ofthis vascular disease could therefore be modulated by antibodies orsmall molecules that bind CtLL polypeptides or that bind to a CtLLbinding partner and trigger or inhibit its activity. In addition,soluble forms of a CtLL polypeptide of the invention would modulatedisease progression by blocking a cellular inhibitory activity of CtLL(as discussed more fully below).

Since many C-type lectins, including OLR1, CLEC1, and CLEC2, areexpressed on macrophages or dendritic cells, the CtLL polypeptides ofthe invention likely play a role in binding to the membrane of apoptoticcells or cell fragments. There is increasing evidence that antigenpresenting cells recognize the exposed phosphatidylserine andphosphatidylcholine lipids on such apoptotic cells and cell fragmentsand take up the fragments for processing and antigen presentation.Current evidence suggests this is a particularly potent way to boostimmune responses to cells dying through this process. Accordingly, aCtLL polypeptide or fragment thereof of the invention would therefore beexpected to modulate the response of antigen presenting cells (APCs) tosuch fragments. A strong immune response would be especially beneficialin the treatment of tumors and cells carrying a pathogen (e.g.,virally-infected cells). Blocking an inhibitory activity of a CtLLpolypeptide using antibodies or soluble fragments of CtLL polypeptideswould be one method of promoting such a response. In contrast, strongresponses to normal cellular components including the oxLDLs themselveswould be expected to play a pathological role in organ specificautoimmune diseases involving cell apoptosis such as diabetes andvascular diseases. Such anti-lipid responses are associated withthrombolytic complications in the PLS (anti-phospholipid syndrome)including that associated with SLE. In some instances promoting anegative signal by activating the ITIM domain of a CtLL polypeptide mayprove beneficial. Antibodies can be developed which stimulate theactivity of a CtLL polypeptide (i.e., stimulate the inhibitory activityof a CtLL polypeptide), such antibodies would be useful in promoting anegative response.

The novel CtLL polypeptides of the present invention have not beendetected in dendritic cells but are detected on monocytes (see Example 8below). In a separate analysis, CtLL polypeptides have been found to bedown-regulated 3-4 fold in a murine asthma model during the onset andincreased severity of the disease (as shown in Example 7 below). Thismay indicate that during inflammation, macrophage processing ofapoptotic cells is downregulated, possibly to protect against excessiveautoantigen presentation. This is consistant with lower CtLL expressionlevels when macrophages are activated in vitro.

In accordance with the invention, any polynucleotide sequence, whichencodes a CtLL polypeptide sequence, can be used to generate recombinantmolecules that can be used to express a CtLL.

A polypeptide includes any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation), and include natural proteins, synthetic or recombinantpolypeptides and peptides as well as a recombinant molecule consistingof a hybrid with one portion, for example, comprising all or part of aCtLL amino acid sequence, or CtLL amino acid sequence and a secondportion being encoded by all or part of a different nucleotide sequence.A polypeptide may comprise L or D amino acids or a combination thereof.D-amino acids have been shown to increase the stability of polypeptidein biological systems by reducing the degradation by proteases.Typically a protein or polypeptide is substantially pure of othercomponents from which it is normally present in nature. The term“substantially pure” or “purified” when referring to a polypeptide,means a polypeptide that is at least 30% free from the proteins andnaturally-occurring organic molecules with which it is naturallyassociated. Preferably the substantially pure polypeptide of theinvention is at least 35-50%; preferably 60-70%; more preferably atleast 75%-90%; and most preferably at least 99% by weight purified fromother naturally occurring molecules. A substantially pure polypeptide ofthe invention can be obtained, for example, by extraction from a naturalsource, by expression of a recombinant polynucleotide encoding thepolypeptide, or by chemically synthesizing the polypeptide. Purity canbe measured by any appropriate method, e.g., chromatography, PAGE, orHPLC analysis.

As used herein a “CtLL polypeptide” means (1) a polypeptide thatcontains or comprises an amino acid sequence as set forth in SEQ IDNO:2, 4, 6, 8, 10, 12, 14, or 16; (2) polypeptides having substantialhomology or substantial identity to the sequences set forth in SEQ IDNO:2, 4, 6, 8, 10, 12, 14, or 16; (3) fragments of the foregoingsequences, including soluble fragments as discussed more fully below;and (4) conservative variants of the foregoing. Examples of preferredfragments of the invention include soluble fragments.

The CtLL polypeptides provided herein have a high degree of homology tomembers of the C-type lectin family and thus have a predicted functionor activity of a C-type lectin polypeptide. Accordingly, the inventionprovides a CtLL polypeptide comprising a sequence as set forth in SEQ IDNO:2, 4, 6, 8, 10, 12, 14, or 16. A CtLL polypeptide can have anactivity characteristic of C-type lectin family members, includingactivities characteristic of OLR1, CLEC1, and/or CLEC2. In oneembodiment, a CtLL polypeptide has carbohydrate-binding or oxidizedlow-density lipoprotein (oxLDL) binding activity. In yet anotherembodiment, a CtLL polypeptide or fragment thereof has agglutinationactivity (e.g., activating or inhibiting agglutination) or immunemodulatory activity. Methods of determining whether a polypeptide of theinvention has a desired activity can be accomplished by assaying thepolypeptide by any of the methods described herein below.

A polypeptide of the invention also encompasses an amino acid sequencethat has a substantial degree of identity or similarity to a sequenceset forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, or 16. Substantiallyidentical sequences can be identified by those of skill in the art ashaving structural domains and/or having biological activity in commonwith a CtLL polypeptide. Methods of determining similarity or identitymay employ computer algorithms such as, e.g., BLAST, FASTA, and thelike.

The phrase “substantially identical,” in the context of two nucleic acidmolecules or polypeptides, refers to sequences or subsequences that haveat least 50%, 60%, preferably 80% or 85%, more preferably 90-95%, andmost preferably 96%, 97%, 98%, or 99% nucleotide or amino acid residueidentity when aligned for maximum correspondence over a comparisonwindow as measured by, for example, a sequence comparison algorithm orby manual alignment and visual inspection. This definition also refersto the complement of a test sequence, which has substantial sequence orsubsequence complementarity when the test sequence has substantialidentity to a reference sequence. A “comparison window,” as used herein,includes reference to a segment of any one of the number of contiguouspositions selected from the group consisting of from 20 to 1800, usuallyabout 50 to 200, more usually about 70 to 150 in which a sequence may becompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection.

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.PILEUP uses a simplification of the progressive alignment method of Feng& Doolittle, J. Mol. Evol. 35:351 (1987), and is similar to the methoddescribed by Higgins & Sharp, CABIOS 5:151 (1989). The multiplealignment procedure begins with the pairwise alignment of the two mostsimilar sequences, producing a cluster of two aligned sequences. Thiscluster is then aligned to the next most related sequence or cluster ofaligned sequences. Two clusters of sequences are aligned by a simpleextension of the pairwise alignment of two individual sequences. Thefinal alignment is achieved by a series of progressive, pairwisealignments. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, asdescribed in Altschul et al., J. Mol. Biol. 215:403 (1990). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (www-ncbi.nlm.nih.gov/). Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy a positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold. These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Extension of the word hits in each direction are halted when:the cumulative alignment score falls off by the quantity X from itsmaximum achieved value; the cumulative score goes to zero or below, dueto the accumulation of one or more negative-scoring residue alignments;or the end of either sequence is reached. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see,Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands. One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.2, more preferably less than about0.01, and most preferably less than about 0.001.

Alternatively, the percent identity of two amino acid or two nucleicacid sequences can be determined by comparing sequence information usingthe GAP computer program, version 6.0 described by Devereux et al.(Nucl. Acids Res. 12:387, 1984) and available from the University ofWisconsin Genetics Computer Group. The preferred default parameters forthe GAP program include: (1) a unary comparison matrix (containing avalue of 1 for identities and 0 for non-identities) for nucleotides, andthe weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas ofPolypeptide Sequence and Structure, National Biomedical ResearchFoundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and anadditional 0.10 penalty for each symbol in each gap; and (3) no penaltyfor end gaps.

One of skill will recognize that individual substitutions, deletions oradditions to a nucleic acid sequence, peptide, or polypeptide sequencethat alters, adds or deletes a single amino acid or a small percentageof amino acids in the encoded sequence is a “conservatively modifiedvariant” where the alteration results in a molecule having substantiallythe same biological activity (e.g., oxLDL binding, carbohydrate bindingactivity, immune modulatory activity, and/or agglutination activity).For example, an alteration that results in the substitution of an aminoacid with a chemically similar amino acid is a conservatively modifiedvariant. Conservative substitution tables providing functionally similaramino acids are known in the art. The following six groups each containamino acids that are conservative substitutions for one another 1)Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W) (see, e.g., Creighton,Proteins (1984)).

One indication that two polynucleotides or polypeptides aresubstantially identical is that the polypeptide encoded by a firstpolynucleotide is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by a second polynucleotide.Another indication that two polynucleotides are substantially identicalis that the two molecules or their complements hybridize to each otherunder stringent conditions.

Polypeptides derived from the CtLL polypeptides of the invention by anytype of alteration (e.g., insertions, deletions, or substitutions ofamino acids; changes in the state of glycosylation of the polypeptide;refolding or isomerization to change its three-dimensional structure orself-association state; and changes to its association with otherpolypeptides or molecules) are also encompassed by the invention.Therefore, the polypeptides provided by the invention includepolypeptides characterized by amino acid sequences similar to those asset forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, or 16, but into whichmodifications are naturally provided or deliberately engineered. Apolypeptide that shares biological activities in common with apolypeptide comprising a sequence as set forth in SEQ ID NO:2, 4, 6, 8,10, 12, 14, or 16, having C-type lectin activity (e.g., oxLDL binding,carbohydrate binding activity, immune modulatory activity, and/oragglutination activity) are encompassed by the invention.

Of particular interest are soluble domains of the CtLL polypeptides ofthe invention. The polypeptides of the invention have a number ofdistinct domains. Table 1 below shows a number of domains associatedwith each of the CtLL polypeptides of the invention comprising SEQ IDNO:2, 4, 6, 8, 10, 12, 14 and 16.

TABLE 1 Transmembrane Domain(s) Soluble Domain(s) C-Lectin LikeDomain(s) SEQ ID NO: (From about residue # to about #) (From aboutresidue # to about #) (From about residue # to about #) 2 42 to 65 1 to41 137 to 159 66 to 136 164 to 182 160 to 163  4 42 to 65 1 to 41 143 to226 66 to 233 6 42 to 65 1 to 41 143 to 227 66 to 232 8 42 to 65 1 to 41137 to 159 66 to 136 160 to 176  10 42 to 65 1 to 41 142 to 247 66 to247 12 42 to 65  1 to 41 142 to 276 66 to 276 14 42 to 65 1 to 41 137 to159 66 to 136 160 to 178  16 42 to 65 1 to 41 137 to 159 66 to 136 160to 176 The domains identified above were predicted using computer algorithmsand/or comparison to known C-type lectin polypeptides. Accordingly, theN- and C-terminal domains may vary by a few amino acids depending uponsuch factors as the type of cell the molecule is expressed in and/or thetypes of proteases present in the expression or purification system. Theidentity of the terminal amino acid can be determined followingexpression by routine peptide sequencing procedures.

The CtLL polypeptides of the invention also have and at least oneimmunoreceptor tyrosine-based inhibitory motif (ITIM). Many receptorsthat mediate positive signaling have cytoplasmic tails containing sitesof tyrosine phosphatase phosphorylation known as immunoreceptortyrosine-based activation motifs (ITAM). A common mechanistic pathwayfor positive signaling involves the activation of tyrosine kinases thatphosphorylate sites on the cytoplasmic domains of the receptors and onother signaling molecules. Once the receptors are phosphorylated,binding sites for signal transduction molecules are created whichinitiate the signaling pathways and activate the cell. The inhibitorypathways involve receptors having immunoreceptor tyrosine basedinhibitory motifs (ITIM) which, like the ITAMs, are phosphorylated bytyrosine kinases. Receptors having ITIM motifs are involved ininhibitory signaling, which block signaling by removing tyrosine fromactivated receptors or signal transduction molecules (Renard et al.,Immun Rev 155:205-221, 1997). ITIMs have the consensus sequenceI/VxYxxL/V (SEQ ID NO:17), and are found in the cytoplasmic portions ofdiverse signal transduction proteins of the immune system, many of whichbelong to the Ig superfamily or to the family of type II dimericC-lectins. Proteins that contain ITIMs include the “killer cell Ig-likereceptors,” or “KIRs,” and some members of the leukocyte Ig-likereceptor or “LIR” family of proteins (Cosman et al., Immunity 7:273-82,1997; Borges et al., J Immunol 159:5192-96, 1997). Signal transductionby an ITIM is believed to downregulate targeted cellular activities,such as expression of cell surface proteins. It is thought that theregulation of complex cellular functions is fine-tuned by the interplayof ITIM-mediated inhibitory signal transduction and activation of thesame functions by the 16-18 amino acid ITAM activator motif. CD22 andFcγRIIb1 also have ITIMs in their cytoplasmic domain and function tosend inhibitory signals that down regulate or inhibit cell function. Ithas been shown that these receptors associate with SHP-1 phosphatase viabinding to the ITIM motifs. Recruitment of the SHP-1 phosphatase by thereceptor appears to be required for intracellular signaling pathwaysthat regulate the inhibitory function of the receptors. Significantly,C-type lectins that are type II membrane proteins having a singleintracellular ITIM motif have also been reported. For example, geneslocalized on human chromosome 12p12-p13 in a region designated as the NKgene complex includes products of the NKG2 complex and CD94, which areinvolved in recognition of MHC class I molecules and in regulation of NKcell activity. Inhibition of cellular functions by NKG2A/B-CD94heterodimers is linked to the presence of ITIMs in the NKG2A/Bintracellular domain (Lazetic, S. C., et al., J. Immunol. 157:4741,1996; Houchins, J. P., et al., J. Immunol. 158:3603, 1997).

Thus, by analogy with other C-type lectin family members having ITIMmotifs, the polypeptides presented in SEQ ID NO:2, 4, 6, 8, 10, 12, 14,or 16 having ITIM motifs, deliver an inhibitory signal via theinteraction of its ITIM with one or more phosphatases, such as tyrosinephosphatases (including SHP-1 tyrosine phosphatase), when the CtLLpolypeptides are bound with an appropriate receptor or natural ligand.Also by analogy with immunoregulatory receptors possessing ITIMs, CtLLfamily members have a regulatory influence on humoral and cell-mediatedimmunity, recognition of MHC class I molecules and in regulation ofimmune cell activity, as well as modulating inflammatory and allergicresponses. Clearly, the immune system activatory and inhibitory signalsmediated by opposing kinases and phosphatases are very important formaintaining balance in the immune system. Systems with a predominance ofactivatory signals will lead to autoimmunity and inflammation. Immunesystems with a predominance of inhibitory signals are less able tochallenge infected cells or cancer cells. Thus, CtLL family members playa role in maintaining balance in the immune system.

The progression of this vascular disease can be modulated by antibodiesor small molecules that bind CtLL polypeptides and trigger or inhibitits activity. In addition, soluble forms of a CtLL polypeptide of theinvention can modulate disease progression by blocking the cellularinhibitory activity of CtLL.

Since many C-type lectins, including OLR1, CLEC1, and CLEC2, areexpressed on macrophages and dendritic cells, the CtLL polypeptides ofthe invention likely play a role in binding to the membrane of apoptoticcells or cell fragments. Although the CtLL polypeptides of the presentinvention have not been detected on dendritic cells, they have beendetected on monocytes. There is increasing evidence that antigenpresenting cells including macrophages recognize the exposedphosphatidylserine and phosphatidylcholine lipids on such cells and takeup the fragments for processing and antigen presentation. Currentevidence suggests this is a particularly potent way to boost immuneresponses to cells dying through this process. Accordingly, the CtLLpolypeptides would therefore be expected to modulate the response ofantigen presenting cells (APCs) to such fragments. Such a strong immuneresponse would be beneficial for treating tumors and pathogen-infectedcells (e.g., virally-infected cells). Blocking the inhibitory activityof the CtLL polypeptides using antibodies or soluble fragments of CtLLpolypeptides would be one method of promoting such a response. Incontrast, strong responses to normal cellular components including theoxLDLs themselves would be expected to play a pathological role in organspecific autoimmune diseases involving cell apoptosis such as diabetesand vascular diseases. Such anti-lipid responses are associated withthrombolytic complications in PLS (anti-phospholipid syndrome) includingthat associated with SLE. In such instances promoting a negative signalby activating the ITIM domain of a CtLL polypeptide may provebeneficial.

Strong responses to normal cellular components including the oxLDLsthemselves would be expected to play a pathological role in organspecific autoimmune diseases involving cell apoptosis such as diabetesand vascular diseases. Such anti-lipid responses are associated withthrombolytic complications in PLS (anti-phospholipid syndrome) includingthat associated with SLE. In such instances promoting a negative signalby activating the ITIM domain of a CtLL polypeptide may provebeneficial.

The protein ligands for the ITIM-containing members of the genomiccluster include MHC molecules for example, HLA-E binding to theheterodimer CD94-NKG2A/B. These molecules down-regulate innate immuneresponses in NK cells and block NK killing of self via their ligandrecognition. Loss of expression of their ligand allows NK cells to lysetumor cells or pathogen infected cells. One feature of these moleculesis that they transit to the cell surface and exert their activities ashetero- or homodimers. Thus, a CtLL polypeptide can bind a proteinligand and block or lower immune responses via HLA-like or MHC-likemolecules potentially as a complex with another molecule in a multimericform. Most likely this response will be mediated through leukocytesother than, or in addition to, NK cells. Thus, a CtLL polypeptide(including soluble fragments thereof) are important in clinical settingsincluding infection and cancer by altering immune system and therecognition of cells as self and non-self. Blocking CtLL function using,for example, a soluble CtLL polypeptide or antibody would boost tumorresponses and responses to pathogen infected cells.

The present invention encompasses the use of various forms of CtLLpolypeptides or domains that have at least one activity selected fromthe group consisting of carbohydrate binding activity, lipoproteinbinding activity (e.g., oxLDL binding activity), agglutinationinhibition activity, cell-cell adhesion activity, capable of binding orinteracting with a proteinaceous ligands, binding or interacting withapoptotic cells, and modulate the activation of tyrosine phosphatases. ACtLL soluble domain is intended to encompass polypeptides comprising allor part of a CtLL polypeptide of the invention but lacking atransmembrane domain. Of particular interest are CtLL soluble domainscharacterized as having (1) identity to and/or biological activity of alectin domain, (2) having carbohydrate-binding activity, and/or (3)having lipoprotein binding activity. In a one embodiment, a solubledomain contains all or part of a CtLL extracellular or cytoplasmicdomain, with or without other domains (e.g., the transmembrane domain),as well as related forms including, but not limited to: (a) fragments,(b) variants, (c) derivatives, (d) fusion polypeptides, and (e)multimeric forms (multimers). The ability of these related forms toinhibit or modulate carbohydrate binding, lipoprotein binding (e.g.,oxidized lipid binding), agglutination, immune system activation, orinflammatory cell activation may be determined in vitro or in vivo byusing methods such as those exemplified below or by using other assaysknown in the art.

A soluble domain (e.g., a fragment of a CtLL polypeptide comprising anextracellular domain) of the invention can provide a dominant-negativeactivity. A soluble domain of the invention (“solCtLL”) includespolypeptides comprising a sequence shown in columns 3 and/or 4 of Table1 and fragments thereof that bind or interact with proteinaceousligands, bind or interact with oxidized lipids, bind or interact withapoptotic cells, or inhibit the activation of tyrosine phosphatases(typically activated by binding of the native CtLL molecule to itsligand), or inhibits a biological activity exhibited by members of theC-type lectin family of polypeptides. As mentioned above, the CtLLpolypeptides of the invention contain an intracellular ITIM motif and sobinding of a CTLL polypeptide comprising an ITIM motif to its ligand(s)is predicted to down-regulate, through the activation of tyrosinephosphatases, activating signals received by the cell in the context ofthe CtLL ligand(s). Accordingly, soluble forms of the ligand-bindingdomain of a CtLL polypeptide would therefore be predicted to increasecellular activation. Preferably the solCtLL comprises an amino acidsequence as set forth in SEQ ID NO:4, 6, 10, or 12 (e.g., a polypeptidecomprising a sequence from about 143 to 226 of SEQ ID NO:4; from about143 to 227 of SEQ ID NO:6; from about 142 to 247 of SEQ ID NO:10; and/orfrom about 142 to 276 of SEQ ID NO:12).

One of skill in the art can assay for activity using the methodsdescribed herein. Such methods measure, for example, carbohydratebinding activity, agglutination inhibition activity, cell-cell adhesionactivity, binding or interaction with proteinaceous ligands, binding orinteraction with oxidized lipids, binding or interaction with apoptoticcells, inhibition of the activation of tyrosine phosphatases, orinhibition of a biological activity exhibited by members of the C-typelectin family of polypeptides. For example, anti-CtLL antibodies, whichneutralize CtLL activity (e.g., binding or interaction withproteinaceous ligands, binding or interaction with oxidized lipids,binding or interaction with apoptotic cells, activation of tyrosinephosphatases, cell-cell adhesion activity, and the like) can be used toassay for similar polypeptides by contacting an anti-CtLL antibody witha polypeptide of interest and determining if the activity associatedwith the polypeptide of interest is neutralized. Similarly, the activityof solCtLL polypeptide may be determined by contacting a cell, tissue,or subject that expresses a native CtLL polypeptide with the solCtLLpolypeptide and measuring a change in, for example, binding orinteraction with proteinaceous ligands, binding or interaction withoxidized lipids, binding or interaction with apoptotic cells, inhibitionof the activation of tyrosine phosphatases, and/or cell-cell adhesionactivity.

The invention provides both full-length and mature forms of CtLLpolypeptides. Full-length polypeptides are those having the completeprimary amino acid sequence of the polypeptide as initially translated.The amino acid sequences of full-length polypeptides can be obtained,for example, by translation of the complete open reading frame (“ORF”)of a cDNA molecule (e.g., SEQ ID NO:1, 3, 5, 7, 9, 11, 13, or 15).Several full-length polypeptides may be encoded by a single geneticlocus if multiple mRNA forms are produced from that locus by alternativesplicing or by the use of multiple translation initiation sites. The“mature form” of a polypeptide refers to a polypeptide that hasundergone post-translational processing steps, if any, such as, forexample, cleavage of the signal sequence or proteolytic cleavage toremove a prodomain. Multiple mature forms of a particular full-lengthpolypeptide may be produced, for example, by imprecise cleavage of thesignal sequence, or by differential regulation of proteases that cleavethe polypeptide. The mature form(s) of such polypeptide may be obtainedby expression, in a suitable mammalian cell or other host cell, of apolynucleotide that encodes the full-length polypeptide. The sequence ofthe mature form of the polypeptide may also be determinable from theamino acid sequence of the full-length form, through identification ofsignal sequences or protease cleavage sites.

The CtLL polypeptides of the invention also include polypeptides thatresult from post-transcriptional or post-translational processing eventssuch as alternate mRNA processing which can yield a truncated butbiologically active polypeptide, for example, a naturally occurringsoluble form of the polypeptide. Also encompassed within the inventionare variations attributable to proteolysis such as differences in the N-or C-termini upon expression in different types of host cells, due toproteolytic removal of one or more terminal amino acids from thepolypeptide (generally from 1-5 terminal amino acids).

A polypeptide of the invention may be prepared by culturing transformedor recombinant host cells under culture conditions suitable to express apolypeptide of the invention. The resulting expressed polypeptide maythen be purified from such culture using known purification processes,such as gel filtration and ion exchange chromatography. The purificationof the polypeptide may also include an affinity column containing agentswhich will bind to the polypeptide; one or more column steps over suchaffinity resins as concanavalin A-agarose, heparin-toyopearl® orCibacrom blue 3GA Sepharose®; one or more steps involving hydrophobicinteraction chromatography using such resins as phenyl ether, butylether, or propyl ether; or immunoaffinity chromatography. Alternatively,a polypeptide of the invention may also be expressed in a form that willfacilitate purification. For example, it may be expressed as a fusionpolypeptide, joined to, for example, maltose binding polypeptide (MBP),glutathione-S-transferase (GST) or thioredoxin (TRX). Kits forexpression and purification of such fusion polypeptides are commerciallyavailable from New England BioLab (Beverly, Mass.), Pharmacia(Piscataway, N.J.), and InVitrogen, respectively. The polypeptide canalso be tagged with an epitope and subsequently purified by using aspecific antibody directed to such epitope. One such epitope (“Flag”) iscommercially available from Kodak (New Haven, Conn.). Finally, one ormore reverse-phase high performance liquid chromatography (RP-HPLC)steps employing hydrophobic RP-HPLC media, e.g., silica gel havingpendant methyl or other aliphatic groups, can be employed to furtherpurify the polypeptide. Some or all of the foregoing purification steps,in various combinations, can also be employed to provide a substantiallyhomogeneous recombinant polypeptide. The polypeptide thus purified issubstantially free of other mammalian polypeptides and is defined inaccordance with the invention as a “substantially purified” polypeptide;such purified polypeptides include antibodies that specifically bind toa CtLL polypeptide, fragment, variant, and the like. A polypeptide ofthe invention may also be expressed as a product of transgenic animals,e.g., as a component of the milk of transgenic cows, goats, pigs, orsheep which are characterized by somatic or germ cells containing apolynucleotide encoding a polypeptide of the invention.

It is also possible to utilize an affinity column such as a monoclonalantibody generated against polypeptides of the invention, toaffinity-purify expressed polypeptides. These polypeptides can beremoved from an affinity column using conventional techniques, e.g., ina high salt elution buffer and then dialyzed into a lower salt bufferfor use or by changing pH or other components depending on the affinitymatrix utilized, or be competitively removed using the naturallyoccurring substrate of the affinity moiety, such as a polypeptidederived from the invention. In this aspect of the invention, proteinsthat bind a polypeptide of the invention (e.g., an anti-CtLL antibody ofthe invention) can be bound to a solid phase support or a similarsubstrate suitable for identifying, separating, or purifying cells thatexpress polypeptides of the invention on their surface. Adherence of,for example, an anti-CtLL antibody of the invention to a solid phasesurface can be accomplished by any means known in the art. For example,magnetic microspheres can be coated with these polypeptide-bindingproteins and held in the incubation vessel through a magnetic field.Suspensions of cell mixtures are contacted with the solid phase that hassuch polypeptide-binding proteins thereon. Anti-CtLL antibodies bindcells having polypeptides of the invention on their surface (e.g., anextracellular domain of CtLL). Unbound cells (e.g., cell lacking andCtLL polypeptide) are washed away from the bound cells. Thisaffinity-binding method is useful for purifying, screening, orseparating such polypeptide-expressing cells from solution. Methods ofreleasing positively selected cells from the solid phase are known inthe art and encompass, for example, the use of enzymes. Such enzymes arepreferably non-toxic and non-injurious to the cells and are preferablydirected to cleaving the cell-surface binding partner. Alternatively,mixtures of cells suspected of containing polypeptide-expressing cellsof the invention are first incubated with a biotinylated bindingpolypeptide of the invention. Incubation periods are typically at leastone hour in duration to ensure sufficient binding to polypeptides of theinvention. The resulting mixture then is passed through a column packedwith avidin-coated beads, whereby the high affinity of biotin for avidinprovides the binding of the cells to the beads. Use of avidin-coatedbeads is known in the art (see, Berenson, et al. J. Cell. Biochem.,10D:239, 1986). Wash of unbound material and the release of the boundcells is performed using conventional methods. Carbohydrate moleculescan also be used to purify cells comprising the polypeptides of theinvention. Carbohydrate molecules that specifically interact with thelectin domain of the polypeptides of the invention can be used to purifythe polypeptides. The polypeptides of the invention will bind to thecarbohydrate via the lectin domain and the cell and/or polypeptide cansubsequently be purified by using techniques known in the art.

A polypeptide of the invention may also be produced by knownconventional chemical synthesis. Methods for constructing thepolypeptides of the invention by synthetic means are known to thoseskilled in the art. The synthetically-constructed polypeptide sequences,by virtue of sharing primary, secondary or tertiary structural and/orconformational characteristics with a native polypeptides may possessbiological properties in common therewith, including biologicalactivity. Thus, the synthesized polypeptides may be employed asbiologically active or immunological substitutes for natural, purifiedpolypeptides in screening of therapeutic compounds, and in immunologicalprocesses for the development of antibodies.

The desired degree of purity depends on the intended use of thepolypeptide. A relatively high degree of purity is desired when thepolypeptide is to be administered in vivo, for example. In such a case,the polypeptides are purified such that no polypeptide bandscorresponding to other polypeptides are detectable upon analysis bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognizedby one skilled in the pertinent field that multiple bands correspondingto the polypeptide can be visualized by SDS-PAGE, due to differentialglycosylation, differential post-translational processing, and the like.Most preferably, the polypeptide of the invention is purified tosubstantial homogeneity, as indicated by a single polypeptide band uponanalysis by SDS-PAGE. The polypeptide band can be visualized by silverstaining, Coomassie blue staining, or (if the polypeptide isradiolabeled) by autoradiography.

Species homologues of CtLL polypeptides and polynucleotides encoding thepolypeptides are also provided by the invention. As used herein, a“species homologue” is a polypeptide or a polynucleotide with adifferent species of origin from that of a given polypeptide orpolynucleotide, but with significant sequence similarity to the givenpolypeptide or polynucleotide. Species homologues may be isolated andidentified by making suitable probes or primers from polynucleotidesencoding the polypeptides provided herein and screening a suitablenucleic acid source from the desired species. One such variant is themurine CtLL, described in the Examples below. Alternatively, homologuesmay be identified by screening a genome database containing sequencesfrom one or more species utilizing a sequence (e.g., nucleic acid oramino acid sequence) of a CtLL molecule of the invention. Such genomedatabases are readily available for a number of species (e.g., on theworld wide web (www) at tigr.org/tdb; genetics.wisc.edu;stanford.edu/˜ball; hiv-web.lan1.gov; ncbi.nlm.nig.gov; ebi.ac.uk; andpasteur.fr/other/biology). The invention also encompasses allelicvariants of CtLL polypeptides and nucleic acids encoding them that arenaturally-occurring alternative forms of such polypeptides andpolynucleotides in which differences in amino acid or nucleotidesequence are attributable to genetic polymorphism.

Intermediate Sequence Search (ISS) can be used to identify closelyrelated as well as distant homologues by connecting two proteins throughone or more intermediate sequences. ISS repetitively uses the results ofthe previous query as new search seeds. Saturated BLAST is a packagethat performs ISS. Starting with a protein sequence, Saturated BLASTruns a BLAST search and identifies representative sequences for the nextgeneration of searches. The procedure is run until convergence or untilsome predefined criteria are met. Saturated BLAST is available on theworld wide web (www) at: bioinformatics.burnham-inst.org/xblast (seealso, Li et al. Bioinformatics 16(12):1105, 2000).

Fragments of the CtLL polypeptides of the invention are encompassed bythe invention and may be in linear form or cyclized using known methods(see, e.g., Saragovi, et al., Bio/Technology 10, 773 (1992); andMcDowell, et al., J. Amer. Chem. Soc. 114:9245 (1992), both of which areincorporated by reference herein). Peptide fragments of CtLLpolypeptides of the invention, and polynucleotides encoding suchfragments include amino acid or nucleotide sequence lengths that are atleast 25% (more preferably at least 50%, 60%, or 70%, and mostpreferably at least 80%) of the length of a CtLL polypeptide orpolynucleotide. Preferably such sequences will have at least 60%sequence identity (more preferably at least 70%-75%, 80%-85%, 90%-95%,at least 97%-97.5%, or at least 99%, and most preferably at least 99.5%)with a CtLL polypeptide or polynucleotide when aligned so as to maximizeoverlap and identity while minimizing sequence gaps. Also included inthe invention are polypeptides, peptide fragments, and polynucleotidesencoding such fragments, that contain or encode a segment preferablycomprising at least 8 to 10, or more preferably at least 20, or stillmore preferably at least 30, or most preferably at least 40 contiguousamino acids. Such polypeptides and fragments may also contain a segmentthat shares at least 70% (at least 75%, 80%-85%, 90%-95%, at least97%-97.5%, or at least 99%, and most preferably at least 99.5%) with anysuch segment of, for example, any of the C-type lectin familypolypeptides, when aligned so as to maximize overlap and identity whileminimizing sequence gaps. Visual inspection, mathematical calculation,or computer algorithms can determine the percent identity.

The invention also provides soluble forms of CtLL polypeptides (solCtLL)comprising certain fragments or domains of the CtLL polypeptides of SEQID Nos:2, 4, 6, 8, 10, 12, 14, and 16. Soluble fragments capable ofbinding or interacting with proteinaceous ligands, binding orinteracting with oxidized lipids, binding or interacting with apoptoticcells, inhibiting the activation of tyrosine phosphatases (typicallyactivated by binding of the native CtLL molecule to its ligand), orinhibition of a biological activity exhibited by members of the C-typelectin family of polypeptides, are of particular interest and include,for example, an amino acid sequence as set forth in Table 1, columns 3and 4. For example, soluble CtLL polypeptides (“solCtLL”) of the presentinvention comprise amino acids: from about residue 1 to about 41 of SEQID NO:2, 4, 6, 8, 10, 12, 14, or 16; from about residue 66 to about 136of SEQ ID NO:2, 8, 14, and 16; from about residue x₁ to about x₂ of SEQID NO:4, wherein x₁ is a residue between and including residues 66 and143 and x₂ is a residue between and including residue 226 and 233 (e.g.,226, 227, 228, 229, etc.); from about residue x₁ to about x₂ of SEQ IDNO:6, wherein x₁ is a residue between and including residues 66 and 143and x₂ is a residue between and including residue 227 and 232 (e.g.,227, 228, 229, 230, etc.); from about residue x₁ to about 247 of SEQ IDNO:10, wherein x₁ is a residue between and including about residues 66and 142; and/or from about residue x₁ to about 276 of SEQ ID NO:12,wherein x₁ is a residue between and including residues 66 and 142. Sucha solCtLL can be secreted from the cell in which it is expressed. A usedherein, the term “between about” or “at about” will be understood toinclude sequences between any such referenced residues of a sequence.For example, “a residue between and including about residue 66 and 143”means residue 66, 67, 68, 69, 70, . . . , 140, 141, 142, or 143”.

Transmembrane regions can be identified using publicly availablecomputer algorithms. The intracellular and transmembrane domains ofpolypeptides of the invention can be identified in accordance with knowntechniques for determination of such domains from sequence information.For example, alignment of the polypeptide sequences of the inventionwith other members of the C-type lectin family of polypeptides havingknown domains will provide information regarding the domains of thepolypeptides of the invention. Accordingly, an alignment and computeralgorithm analysis of SEQ ID Nos:2, 4, 6, 8, 10, 12, 14, and 16indicates a transmembrane domain includes about amino acids 42 to 65 ofSEQ ID Nos:2, 4, 6, 8, 10, 12, 14, and 16; includes about amino acids137 to 159 of SEQ ID Nos:2, 8, 14, and 16; and includes about aminoacids 164 to 182 of SEQ ID NO:2. One of skill in the art will recognizethat slight modifications in the range of sequences of a particulardomain can be made without affecting the molecule's biological activity.Accordingly, changes in the identified sequences and domains of theinvention by 1, 2, 3, 4, or 5 to 10 amino acids in either direction ofthe particular domain are encompassed by the present invention.

In another aspect of the invention, a polypeptide may comprise variouscombinations of C-type lectin polypeptide domains, such one or morelectin domains linked to a lectin domain of the present invention (e.g.,from about residue x₁ to about x₂ of SEQ ID NO:4, wherein x₁ is aresidue between and including residues 66 and 143 and x₂ is a residuebetween and including residue 226 and 233). Accordingly, polypeptides ofthe invention and polynucleotides include those comprising or encodingtwo or more copies of a domain such as the lectin domain of the presentinvention, a lectin domain of the present invention and C-type lectindomain of another C-type lectin polypeptide, or one or more solCtLLpolypeptides, these domains may be presented in any order within suchpolypeptides. For example, the invention provides a polypeptidecomprising Z₁-X-Z₂, wherein Z₁ and Z₂ are each individually a solCtLLpolypeptide (as described above), and X is a peptide linker.Accordingly, the invention includes recombinant polypeptides and thepolynucleotides encoding the polypeptides wherein the recombinantpolypeptides are “chimeric polypeptides” or “fusion polypeptides” andcomprise a solCtLL sequence as set forth above operatively linked to asecond polypeptide. The second polypeptide can be any polypeptide ofinterest having an activity or function independent of, or related to,the function of a CtLL polypeptide. For example, the second polypeptidecan be a domain of a related but distinct member of the C-type lectinfamily of polypeptides such as, for example, an extracellular,cytoplasmic, or transmembrane domain of a C-type lectin polypeptide. Theterm “operatively linked” is intended to indicate that the CtLL sequenceand the second polypeptide sequence are fused in-frame to each other.The second polypeptide can be fused to the N-terminus or C-terminus of aCtLL or solCtLL sequence as set forth in FIG. 1 or as set forth above.For example, in one embodiment the fusion polypeptide is a GST-CtLL orGST-solCtLL fusion polypeptide in which a CtLL or solCtLL polypeptide isfused to the C-terminus of a GST sequence. Such fusion polypeptides canfacilitate the purification of recombinant CtLL and solCtLL polypeptidesand fragments. In another embodiment, the fusion polypeptide comprises aCtLL or solCtLL sequence having a heterologous signal sequence at itsN-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion of a CtLL or solCtLL polypeptide can beincreased through use of a heterologous signal sequence. As anotherexample, a CtLL polypeptide, solCtLL, or fragments thereof may be fusedto a hexa-histidine tag to facilitate purification of bacteriallyexpressed protein, or to a hemagglutinin tag to facilitate purificationof protein expressed in eukaryotic cells. Further, fusion polypeptidescan comprise, for example, poly-His or the antigenic identificationpeptides described in U.S. Pat. No. 5,011,912 and in Hopp et al.,Bio/Technology 6:1204, 1988. One such peptide is the FLAG® peptide,which is highly antigenic and provides an epitope reversibly bound by aspecific monoclonal antibody, enabling rapid assay and facilepurification of an expressed recombinant polypeptide. A murine hybridomadesignated 4E11 produces a monoclonal antibody that binds the FLAG®peptide in the presence of certain divalent metal cations, as describedin U.S. Pat. No. 5,011,912, hereby incorporated by reference. The 4E11hybridoma cell line has been deposited with the ATCC under accession no.HB9259. Monoclonal antibodies that bind the FLAG® peptide are availablefrom Eastman Kodak Co., Scientific Imaging Systems Division, New Haven,Conn.

Encompassed by the invention are oligomers that comprise a CtLLpolypeptide or solCtLL polypeptide. Oligomers that can be in the form ofcovalently linked or non-covalently-linked multimers, including dimers,trimers, or higher oligomers. In one aspect of the invention, theoligomers maintain the binding ability or biological activity of thepolypeptide components and provide therefor, bivalent, trivalent, andthe like, binding or catalytic sites. In another embodiment, theinvention is directed to oligomers comprising multiple polypeptidesjoined via covalent or non-covalent interactions linked to or betweenpeptide moieties fused to the polypeptides. Such peptides can be peptidelinkers (spacers), or peptides that have the property of promotingoligomerization. Leucine zippers and certain polypeptides derived fromantibodies are among the peptides that can promote oligomerization ofthe polypeptides attached thereto, as described in more detail below.

Peptide linkers can be used between two or more CtLL or solCtLLpolypeptides. Typically a peptide linker moiety is chosen to optimizethe biological activity of the polypeptide comprising a CtLL or solCtLLsequence and can be determined empirically without undueexperimentation. The linker moiety should be long enough and flexibleenough to allow a CtLL or solCtLL polypeptide to freely interact with asubstrate or ligand. The preferred linker moiety is a peptide betweenabout one and 30 amino acid residues in length, preferably between abouttwo and 15 amino acid residues. Preferred linker moieties are -Gly-Gly-,GGGGS (SEQ ID NO:18), (GGGGS)_(n) (SEQ ID NO:18), GKSSGSGSESKS (SEQ IDNO:19), GSTSGSGKSSEGKG (SEQ ID NO:20), GSTSGSGKSSEGSGSTKG (SEQ IDNO:21), GSTSGSGKPGSGEGSTKG (SEQ ID NO:22), or EGKSSGSGSESKEF (SEQ IDNO:23). Linking moieties are described, for example, in Huston, J. S.,et al., PNAS 85:5879 (1988), Whitlow, M., et al., Protein Engineering6:989 (1993), and Newton, D. L., et al., Biochemistry 35:545 (1996).Other suitable peptide linkers are those described in U.S. Pat. Nos.4,751,180 and 4,935,233, which are hereby incorporated by reference. ADNA sequence encoding a desired peptide linker can be inserted between,and in the same reading frame as, a DNA sequences encoding a CtLLpolypeptide or fragment thereof (e.g., a solCtLL), using any suitableconventional technique. For example, a chemically synthesizedoligonucleotide encoding the linker can be ligated between thesequences. In particular embodiments, a fusion polypeptide comprisesfrom two to four solCtLL polypeptides, separated by peptide linkers.

In embodiments where variants of a CtLL polypeptide are constructed toinclude a membrane-spanning domain, they will form a Type II membranepolypeptide. In such embodiments, it is preferable to link the fusionpartner to the C-terminus of the CtLL or solCtLL polypeptide.Alternatively, the membrane-spanning polypeptides can be fused withknown extracellular receptor domain polypeptides, for which the ligandis also known. Such fusion polypeptides can then be manipulated tocontrol an intracellular signaling pathways triggered by the bound CtLLpolypeptide. Polypeptides that span the cell membrane can also be fusedwith agonists or antagonists of cell-surface receptors, or cellularadhesion molecules to further modulate CtLL intracellular effects. Inanother aspect of the invention, interleukins can be situated betweenthe CtLL polypeptide fragment and other fusion polypeptide domains.

The CtLL polypeptides and solCtLL polypeptides of the invention can alsoinclude a localization sequence to direct the polypeptide to particularcellular sites by fusion to appropriate organellar targeting signals orlocalized host proteins. A polynucleotide encoding a localizationsequence (signal sequence), can be ligated or fused at the 5′ terminusof a polynucleotide encoding a CtLL or solCtLL polypeptide such that thelocalization peptide comprising the localization sequence is located atthe amino terminal (5′) end of the resulting fusion polypeptide(polynucleotide). In eukaryotes, the localization peptide functions totransport a polypeptide across the endoplasmic reticulum. The secretorypolypeptide is then transported through the Golgi apparatus, intosecretory vesicles and into the extracellular space or the externalenvironment. Localization (signal) peptides include pre-pro peptidesthat contain a proteolytic enzyme recognition site.

The localization peptide can be a nuclear-, an endoplasmic reticulum-, aperoxisome-, or a mitochondrial-localization sequence, or a localizedprotein. Localization peptide can comprise targeting sequences that aredescribed, for example, in “Protein Targeting”, chapter 35 of Stryer,L., Biochemistry (4th ed.). W. H. Freeman, 1995. Some importantlocalization sequences include those targeting the nucleus (e.g., KKKRK(SEQ ID NO:24)), mitochondria (MLRTSSLFTRRVQPSLFRNI LRLQST (SEQ IDNO:25)), endoplasmic reticulum (KDEL (SEQ ID NO:26)), peroxisome (SKF),plasma membrane (CAAX (SEQ ID NO:27), CC, CXC, or CCXX (SEQ ID NO:28)),cytoplasmic side of plasma membrane (fusion to SNAP-25), or the Golgiapparatus (fusion to furin).

In another embodiment, a polypeptide of the invention or fragmentsthereof may be fused to carrier molecules such as immunoglobulins for avariety of purposes including increasing the valency of polypeptidebinding sites. As an example, a fragment (e.g., solCtLL) of apolypeptide of the invention may be fused through a peptide linker tothe Fc portion of an immunoglobulin. For a bivalent form of thepolypeptide, such a fusion could be to the Fc portion of an IgGmolecule. Other immunoglobulin isotypes may also be used to generatesuch fusions. For example, a polypeptide-IgM fusion would generate adecavalent form of the polypeptide of the invention. In one embodiment,the invention provides a fusion polypeptide having an Fc polypeptidedomain and a CtLL or solCtLL polypeptide sequence.

The term “Fc polypeptide” as used herein includes native and muteinforms of polypeptides made up of the Fc region of an antibody comprisingany or all of the CH domains of the Fc region. Truncated forms of suchpolypeptides containing the hinge region that promotes dimerization arealso included. Preferred polypeptides comprise an Fc polypeptide derivedfrom a human IgG1 antibody. As one alternative, an oligomer is preparedusing polypeptides derived from immunoglobulins. Preparation of fusionpolypeptides comprising certain heterologous polypeptides fused tovarious portions of antibody-derived polypeptides (including the Fcdomain) has been described, e.g., by Ashkenazi et al. (PNAS USA88:10535, 1991); Byrn et al. (Nature 344:677, 1990); and Hollenbaugh andAruffo (“Construction of Immunoglobulin Fusion Polypeptides”, in CurrentProtocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992).Methods for preparation and use of immunoglobulin-based oligomers areknown in the art. One embodiment of the invention is directed to a dimercomprising two fusion polypeptides created by fusing a polypeptide ofthe invention to an Fc polypeptide derived from an antibody. A genefusion encoding the polypeptide/Fc fusion polypeptide is inserted intoan appropriate expression vector. Polypeptide/Fc fusion polypeptides areexpressed in host cells transformed or transfected with the recombinantexpression vector or recombinant polynucleotide encoding the fusionpolypeptide, and allowed to assemble much like antibody molecules,whereupon interchain disulfide bonds form between the Fc moieties toyield divalent molecules. One suitable Fc polypeptide, described in PCTapplication WO 93/10151 (hereby incorporated by reference), is a singlechain polypeptide extending from the N-terminal hinge region to thenative C-terminus of the Fc region of a human IgG1 antibody. Anotheruseful Fc polypeptide is the Fc mutein described in U.S. Pat. No.5,457,035 and in Baum et al., (EMBO J. 13:3992, 1994) incorporatedherein by reference. The amino acid sequence of this mutein is identicalto that of the native Fc sequence presented in WO 93/10151, except thatamino acid 19 has been changed from Leu to Ala, amino acid 20 has beenchanged from Leu to Glu, and amino acid 22 has been changed from Gly toAla. The mutein exhibits reduced affinity for Fc receptors. Theabove-described fusion polypeptides comprising Fc moieties (andoligomers formed therefrom) offer the advantage of facile purificationby affinity chromatography over Polypeptide A or Polypeptide G columns.In other embodiments, the polypeptides of the invention can besubstituted for the variable portion of an antibody heavy or lightchain. If fusion polypeptides are made with both heavy and light chainsof an antibody, it is possible to form an oligomer with as many as fourCtLL or solCtLL polypeptides or fragments thereof.

Another method for preparing the oligomers of the invention involves useof a leucine zipper. Leucine zipper domains are peptides that promoteoligomerization (dimers and trimers) of the polypeptides in which theyare found. Leucine zippers were originally identified in severalDNA-binding polypeptides (Landschulz et al., Science 240:1759, 1988),and have since been found in a variety of different polypeptides. Thezipper domain comprises a repetitive heptad repeat, often with four orfive leucine residues interspersed with other amino acids.

A chimeric or fusion polypeptide of the invention can be produced bystandard recombinant DNA techniques. In one embodiment, polynucleotidefragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample, by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primersthat give rise to complementary overhangs between two consecutive genefragments that can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide).

The invention further includes polypeptides with or without associatednative-pattern glycosylation. Polypeptides expressed in yeast ormammalian expression systems (e.g., COS-1 or CHO cells) can be similarto or significantly different from a native polypeptide in molecularweight and glycosylation pattern, depending upon the choice ofexpression system. Expression of polypeptides of the invention inbacterial expression systems, such as E. coli, provides non-glycosylatedmolecules. Further, a given preparation can include multipledifferentially glycosylated species of the polypeptide. Glycosyl groupscan be removed through conventional methods, in particular thoseutilizing glycopeptidase.

In another embodiment, modifications in the polypeptide orpolynucleotide can be made using known techniques. Modifications ofinterest in the polypeptide sequences may include the alteration,substitution, replacement, insertion, or deletion of a selected aminoacid residue in the coding sequence. For example, one or more of thecysteine residues may be deleted or replaced with another amino acid toalter the conformation of the molecule, an alteration which may involvepreventing formation of incorrect intramolecular disulfide bridges uponfolding or renaturation. Techniques for such alteration, substitution,replacement, insertion, or deletion are known to those skilled in theart (see, e.g., U.S. Pat. No. 4,518,584). As another example,N-glycosylation sites in a polypeptide's extracellular domain can bemodified to preclude glycosylation, allowing expression of a reducedcarbohydrate analog in mammalian and yeast expression systems.N-glycosylation sites in eukaryotic polypeptides are characterized by anamino acid triplet Asn-X-Y, wherein X is any amino acid except Pro, andY is Ser or Thr. Appropriate substitutions, additions, or deletions tothe nucleotide sequence encoding these triplets will result inprevention of attachment of carbohydrate residues at the Asn side chain.Alteration of a single nucleotide, chosen so that Asn is replaced by adifferent amino acid, for example, is sufficient to inactivate anN-glycosylation site. Alternatively, the Ser or Thr can by replaced withanother amino acid, such as Ala. Known procedures for inactivatingN-glycosylation sites in polypeptides include those described in U.S.Pat. No. 5,071,972 and EP 276,846, hereby incorporated by reference. N91and N101 of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, and 16 are putativeglycosylation sites. One of skill in the art can identify the tripletresidues corresponding to the glycosylation site.

Additional variants within the scope of the invention includepolypeptides that can be modified to create derivatives thereof byforming covalent or aggregative conjugates with other chemical moieties,such as glycosyl groups, lipids, phosphate, acetyl groups and the like.Covalent derivatives can be prepared by linking the chemical moieties tofunctional groups on amino acid side chains or at the N-terminus orC-terminus of a polypeptide. Conjugates comprising diagnostic(detectable) or therapeutic agents attached thereto are contemplatedherein. Preferably, such alteration, substitution, replacement,insertion or deletion retains the desired activity of the polypeptide.

The invention also provides polynucleotides encoding CtLL and solCtLLpolypeptides. The term “polynucleotide” refers to a polymeric form ofnucleotides of at least 10 bases in length. The nucleotides can beribonucleotides, deoxyribonucleotides, or modified forms of either typeof nucleotide. The term includes single and double stranded forms of DNAor RNA. DNA includes, for example, cDNA, genomic DNA, chemicallysynthesized DNA, DNA amplified by PCR, and combinations thereof. Thepolynucleotides of the invention include full-length genes and cDNAmolecules as well as a combination of fragments thereof. Thepolynucleotides of the invention are preferentially derived from humansources, but the invention includes those derived from non-human species(e.g., mus musculus), as well.

By “isolated polynucleotide” is meant a polynucleotide that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (one on the 5′ end and one on the 3′ end) inthe naturally occurring genome of the organism from which it is derived.The term therefore includes, for example, a recombinant polynucleotidemolecule, which is incorporated into a vector, e.g., an expressionvector; into an autonomously replicating plasmid or virus; or into thegenomic DNA of a prokaryote or eukaryote, or which exists as a separatemolecule (e.g., a cDNA) independent of other sequences.

A CtLL polynucleotide of the invention (1) encodes a polypeptidecomprising a sequence as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14,or 16; (2) has a sequence as set forth in SEQ ID NO:1, 3, 5, 7, 9, 11,13, or 15; (3) has sequences complementary to a sequence as set forth inSEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15; (4) includes fragments of SEQID NO: 1, 3, 5, 7, 9, 11, 13, or 15 or a complement that specificallyhybridizes to the polynucleotide of (2) or (3) under moderate to highlystringent conditions; and (5) includes polynucleotides of (1), (2), (3),or (4) wherein T can also be U (e.g., RNA sequences). Examples of CtLLpolynucleotide fragments of the invention include the nucleotidesequence corresponding to a solCtLL polypeptide. A solCtLLpolynucleotide encoding a solCtLL polypeptide comprise a sequenceselected from the group consisting of: from about nucleotide 1 to aboutnucleotide 123 of SEQ ID NO:1, 3, or 13; from about nucleotide 196 toabout nucleotide 408 of SEQ ID NO:1 or 13; from about nucleotide 478 to489 of SEQ ID NO:1; from about nucleotide x₃ to about nucleotide x₄ ofSEQ ID NO:3, wherein x₃ is a nucleotide from (and including) about 196to 427 and x₄ is a nucleotide from (and including) about 678 to 699,preferably from about 427 to 678 of SEQ ID NO:3; from about nucleotide37 to about nucleotide 159 of SEQ ID NO:5; from about nucleotide x₃ toabout nucleotide x₄ of SEQ ID NO:5, wherein x₃ is a nucleotide from (andincluding) about 232 to 463 and x₄ is a nucleotide from (and including)about 717 to 732, preferably from about 463 to 717 of SEQ ID NO:5; fromabout nucleotide 49 to about nucleotide 171 of SEQ ID NO:7 or 11; fromabout nucleotide 244 to about nucleotide 456 of SEQ ID NO:7; from aboutnucleotide 526 to about nucleotide 576 of SEQ ID NO:7; from aboutnucleotide 101 to about nucleotide 223 of SEQ ID NO:9; from aboutnucleotide x₃ to about nucleotide 841 of SEQ ID NO:9, wherein x₃ is anucleotide from (and including) about 296 to 524, preferably from about524 to 841 of SEQ ID NO:9; from about nucleotide x₃ to about nucleotide876 of SEQ ID NO:11, wherein x₃ is a nucleotide from (and including)about 244 to 475, preferably from about 475 to 876 of SEQ ID NO:11; fromabout nucleotide 478 to about nucleotide 534 of SEQ ID NO:13; from aboutnucleotide 164 to about nucleotide 286 of SEQ ID NO:15; from aboutnucleotide 359 to about nucleotide 571 of SEQ ID NO:15; and from aboutnucleotide 641 to about nucleotide 690 of SEQ ID NO:15. Also encompassedby the invention are homologues of a CtLL polynucleotide of theinvention. These polynucleotides can be identified in several ways,including isolation of genomic or cDNA molecules from a suitable source,or computer searches of available sequence databases. Oligonucleotidesor polynucleotides corresponding to the amino acid sequences describedherein can be used as probes or primers for the isolation ofpolynucleotide homologues or as query sequences for database searches.Degenerate oligonucleotide sequences can be obtained by“back-translation” from an amino acid sequence of the invention. Thepolymerase chain reaction (PCR) procedure can be employed to isolate andamplify a DNA sequence encoding a CtLL polypeptide or a desiredcombination of CtLL polypeptide fragments. Oligonucleotides that definethe desired termini of a target DNA molecule are employed as 5′ and 3′primers. Accordingly, fragments of the polynucleotides of the inventionare useful as probes and primers to identify or amplify related sequenceor obtain full-length sequences of a CtLL of the invention. Theoligonucleotides can additionally contain recognition sites forrestriction endonucleases, to facilitate insertion of the amplifiedcombination of DNA fragments into an expression vector. PCR techniquesare known in the art (see, e.g., PCR Protocols: A Guide to Methods andApplications, Innis et. al., eds., Academic Press, Inc. (1990)).

The invention also includes polynucleotides and oligonucleotides thathybridize under reduced stringency conditions, more preferablymoderately stringent conditions, and most preferably highly stringentconditions, to CtLL polynucleotides described herein. The basicparameters affecting the choice of hybridization conditions and guidancefor devising suitable conditions are set forth by Sambrook, J., E. F.Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters9 and 11; and Current Protocols in Molecular Biology, 1995, Ausubel etal., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4,incorporated herein by reference), and can be readily determined bythose having ordinary skill in the art based on, for example, the lengthand/or base composition of the polynucleotide. One way of achievingmoderately stringent conditions involves the use of a prewashingsolution containing 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridizationbuffer of about 50% formamide, 6×SSC, and a hybridization temperature ofabout 55° C. (or other similar hybridization solutions, such as onecontaining about 50% formamide, with a hybridization temperature ofabout 42° C.), and washing conditions of about 60° C., in 0.5×SSC, 0.1%SDS. Generally, highly stringent conditions are defined as hybridizationconditions as above, but with washing at approximately 68° C., 0.2×SSC,0.1% SDS. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA,pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCl and 15 mM sodiumcitrate) in the hybridization and wash buffers; washes are performed for15 minutes after hybridization is complete. It should be understood thatthe wash temperature and wash salt concentration can be adjusted asnecessary to achieve a desired degree of stringency by applying thebasic principles that govern hybridization reactions and duplexstability, as known to those skilled in the art and described furtherbelow (see, e.g., Sambrook et al., 1989). When hybridizing a nucleicacid to a target polynucleotide of unknown sequence, the hybrid lengthis assumed to be that of the hybridizing nucleic acid. When nucleicacids of known sequence are hybridized, the hybrid length can bedetermined by aligning the sequences of the nucleic acids andidentifying the region or regions of optimal sequence complementarity.The hybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5 to 10° C. less than the meltingtemperature (T_(m)) of the hybrid, where T_(m) is determined accordingto the following equations. For hybrids less than 18 base pairs inlength, T_(m) (° C.)=2(# of A+T bases)+4(# of G+C bases). For hybridsabove 18 base pairs in T_(m) (° C.)=81.5+16.6(log₁₀ [Na⁺])+0.41(%G+C)−(600/N), where N is the number of bases in the hybrid, and [Na⁺] isthe concentration of sodium ions in the hybridization buffer ([Na⁺] for1×SSC=0.165M). Preferably, each such hybridizing nucleic acid has alength that is at least 25% (more preferably at least 50%, 60%, or 70%,and most preferably at least 80%) of the length of a polynucleotide ofthe invention to which it hybridizes, and has at least 60% sequenceidentity (more preferably at least 70%, 75%, 80%, 85%, 90%, 95%, 97.5%,or at least 99%, and most preferably at least 99.5%) with apolynucleotide of the invention to which it hybridizes.

“Conservatively modified variants” applies to both polypeptide andpolynucleotide. With respect to particular polynucleotide,conservatively modified variants refer to codons in the polynucleotidewhich encode identical or essentially identical amino acids. Because ofthe degeneracy of the genetic code, a large number of functionallyidentical polynucleotides encode any given protein. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such variations are “silent variations,” whichare, one species of conservatively modified variations. Everypolynucleotide sequence herein that encodes a polypeptide also describesevery possible silent variation of the nucleic acid. One of skill willrecognize that each codon in a polynucleotide (except AUG, which isordinarily the only codon for methionine) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid that encodes a polypeptide is implicit in each describedsequence.

The invention also provides methodology for analysis of polynucleotidesof the invention, or fragments thereof, on “DNA chips” as described inHacia et al., Nature Genetics, 14:441-447 (1996). For example,high-density arrays of oligonucleotides comprising a sequence as setforth in SEQ ID NO:1, or a variant or mutant thereof, are applied andimmobilized to the chip and can be used to detect sequence variations ina population. Polynucleotides in a test sample are hybridized to theimmobilized oligonucleotides. The hybridization profile of the targetpolynucleotide to the immobilized probe is quantitated and compared to areference profile. The resulting genetic information can be used inmolecular diagnosis. The density of oligonucleotides on DNA chips can bemodified as needed.

The invention also provides genes corresponding to the polynucleotidesdisclosed herein. “Corresponding gene regions” are the regions of thegenome that are transcribed to produce the mRNAs from which cDNAmolecules are derived and may include contiguous regions of the genomenecessary for the regulated expression of such genes. Correspondinggenes may therefore include but are not limited to coding sequences, 5′and 3′ untranslated regions, alternatively spliced exons, introns,promoters, enhancers, and silencer or suppressor elements. Thecorresponding genes can be isolated in accordance with known methodsusing the sequence information disclosed herein. Such methods includethe preparation of probes or primers from the disclosed sequenceinformation for identification and/or amplification of genes inappropriate genomic libraries or other sources of genomic materials.

Expression, isolation, and purification of the polypeptides andfragments of the invention can be accomplished by any suitabletechnique, including but not limited to the following methods.

The isolated polynucleotides of the invention may be operably linked toan expression control sequence such as the pMT2 or pED expressionvectors disclosed in Kaufman et al., Nucleic Acids Res. 19:4485 (1991);and Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, N.Y.,(1985, and Supplements), in order to produce a polypeptide of theinvention recombinantly. Many suitable expression control sequences areknown in the art. General methods of expressing recombinant polypeptidesare also known and are exemplified in R. Kaufman, Methods in Enzymology185:537 (1990). As defined herein “operably linked” means that anisolated polynucleotide of the invention and an expression controlsequence are situated within a vector or cell in such a way that thepolypeptide encoded by the polynucleotide is expressed by a host cellwhich has been transformed (transfected) with the vector orpolynucleotide operably linked to the control sequence.

In addition, a sequence encoding an appropriate localization (signal)peptide (native or heterologous) can be incorporated into expressionvectors. The choice of signal peptide or leader can depend on factorssuch as the type of host cells in which the recombinant polypeptide isto be produced. Examples of heterologous localization (signal) peptidesthat are functional in mammalian host cells include the signal sequencefor interleukin (IL)-7 (see, U.S. Pat. No. 4,965,195); the signalsequence for IL-2 receptor (see, Cosman et al., Nature 312:768, 1984);the IL-4 receptor signal peptide (see, EP 367,566); the type I IL-1receptor signal peptide (see, U.S. Pat. No. 4,968,607); and the type IIIL-1 receptor signal peptide (see, EP 460,846). A signal peptide that isfunctional in the intended host cells promotes extracellular secretionof the polypeptide. The signal peptide is cleaved from the polypeptideupon secretion of a polypeptide from the cell. A polypeptide preparationcan include a mixture of polypeptide molecules having differentN-terminal amino acids, resulting from cleavage of the signal peptide atmore than one site.

Established methods for introducing DNA into mammalian cells have beendescribed (Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp.15-69). Additional protocols using commercially available reagents, suchas Lipofectamine or Lipofectamine-Plus lipid reagent (Gibco/BRL), can beused to transfect cells (Feigner et al., Proc. Natl. Acad. Sci. USA84:7413, 1987). In addition, electroporation can be used to transfectmammalian cells using conventional procedures, such as those in Sambrooket al. (Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1-3, ColdSpring Harbor Laboratory Press, 1989). Selection of stable transformantscan be performed using methods known in the art, such as, for example,resistance to cytotoxic drugs. Kaufman et al., Meth. in Enzymology185:487, 1990, describes several selection schemes, such asdihydrofolate reductase (DHFR) resistance. A suitable strain for DHFRselection can be CHO strain DX-B11, which is deficient in DHFR (Urlaubet al., Proc. Natl. Acad. Sci. USA 77:4216, 1980). A plasmid expressingthe DHFR cDNA can be introduced into strain DX-B11, and only cells thatcontain the plasmid can grow in the appropriate selective media. Otherexamples of selectable markers that can be incorporated into anexpression vector include cDNAs conferring resistance to antibiotics,such as G418 and hygromycin B. Cells harboring the vector are selectedon the basis of resistance to these compounds.

Alternatively, gene products can be obtained via homologousrecombination, or “gene targeting” techniques. Such techniques employthe introduction of exogenous transcription control elements (such asthe CMV promoter or the like) in a particular predetermined site on thegenome, to induce expression of an endogenous CtLL of the invention. Thelocation of integration into a host chromosome or genome can be easilydetermined by one of skill in the art, given the known location andsequence of the gene. In another embodiment, the invention alsocontemplates the introduction of exogenous transcriptional controlelements in conjunction with an amplifiable gene, to produce increasedamounts of the gene product. The practice of homologous recombination orgene targeting is explained by Schimke, et al. “Amplification of Genesin Somatic Mammalian cells,” Methods in Enzymology 151:85 (1987), and byCapecchi, et al., “The New Mouse Genetics: Altering the Genome by GeneTargeting,” TIG 5:70 (1989).

Suitable host cells for expression of the polypeptide include eukaryoticand prokaryotic cells. Mammalian host cells include, for example, theCOS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinesehamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell lines, theCV1/EBNA cell line derived from the African green monkey kidney cellline CV1 (ATCC CCL 70) (see, McMahan et al. EMBO J. 10: 2821, 1991),human kidney 293 cells, human epidermal A431 cells, human Colo205 cells,other transformed primate cell lines, normal diploid cells, cell strainsderived from in vitro culture of primary tissue, primary explants,HL-60, U937, HaK or Jurkat cells. Alternatively, it may be possible toproduce the polypeptide in lower eukaryotes such as yeast or inprokaryotes such as bacteria. Potentially suitable yeast strains includeSaccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromycesstrains, Candida, or any yeast strain capable of expressing heterologouspolypeptides. Potentially suitable bacterial strains include, forexample, Escherichia coli, Bacillus subtilis, Salmonella typhimurium, orany bacterial strain capable of expressing heterologous polypeptides. Ifthe polypeptide is made in yeast or bacteria, it may be necessary tomodify the polypeptide produced therein, for example by phosphorylationor glycosylation of the appropriate sites, in order to obtain thefunctional polypeptide. Such covalent attachments may be accomplishedusing known chemical or enzymatic methods. The polypeptide may also beproduced by operably linking a polynucleotide of the invention tosuitable control sequences in one or more insect expression vectors, andemploying an insect expression system. Materials and methods forbaculovirus/insect cell expression systems are commercially available inkit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBac®kit), as well as methods described in Summers and Smith, TexasAgricultural Experiment Station Bulletin No. 1555 (1987), and Luckow andSummers, Bio/Technology 6:47 (1988), incorporated herein by reference.Cell-free translation systems could also be employed to producepolypeptides using RNAs derived from nucleic acid constructs disclosedherein. A host cell that comprises an isolated polynucleotide of theinvention, preferably operably linked to at least one expression controlsequence, is a “recombinant host cell”.

In some instances it may be advantageous to suppress CtLL expression orto inhibit the interaction of CtLL with a target cell or ligand. Forinstance, suppression of a CtLL polynucleotide sequence may alleviatedamage associated with over expression or excess CtLL activity in theimmune system and would be beneficial in the activation of the immunesystem for the treatment of tumors and cells carrying a pathogen (e.g.,virally-infected cells). Blocking an inhibitory activity of a CtLLpolypeptide using antisense or ribozyme technology to reduce expressionof a CtLL polypeptides would be one method of promoting such a response.Other diseases and disorders where modulation of CtLL polypeptideexpression or activity include chronic rejection of transplanted tissuesand organs, or in athersclerosis and inflammatory responses mediatedwith plaque formation in vascular tissue. CtLL expression can besuppressed by administration of CtLL antisense oligonucleotides.

In contrast, strong responses to normal cellular components includingthe oxidized lipids themselves would be expected to play a pathologicalrole. Accordingly, an activator of CtLL activity or expression can playa role in the prevention of autoimmune diseases involving cell apoptosissuch as diabetes and vascular diseases. For example, activating theexpression or activity of CtLL would increase the inhibitory activitythereby reducing the immune systems response to such non-self ordiseased cells.

Due to the identity that the CtLL polypeptide have to other C-typelectins the CtLL polypeptides may also play a role in pathogenesis dueto infection. For example, pathogenic Mycobacteria, including M.tuberculosis, colonize in activated macrophages. The attachment of suchpathogens to the activated macrophages is a preliminary step inpathogenesis. Preventing this interaction may provide an importantapproach for blocking the colonization of host macrophages by pathogenicbacteria or parasites. In another embodiment, plaque formationassociated with athersclerosis may be prevented by preventing theinteraction of CtLL with its cognate by using antibodies to CtLL or bythe administration of a solCtLL that binds the cognate and preventsinteraction of the cognate with the naturally occurring CtLL.

In a one embodiment, a soluble fragment of CtLL consisting primarily ofthe extracellular region binding domain may be used as an inhibitor ofnative CtLL or in the prevention of interaction of CtLL with a cognate(e.g., an oxidized lipid). The structural integrity of the isolatedextracellular domain would be maintained by the intrachain disulfidebonds. When administered, the soluble binding domain can blockCtLL-target cell interactions by competing with native CtLL for ligandson the surface of the target cell.

CtLL may be exploited for the purposes of targeted drug delivery.Anti-pathogen and anti-parasite therapies are hampered by thesequestering of the pathogenic bacteria or parasites within infectedcell types (e.g., macrophages), restricting the bioavailability ofpotentially useful drugs. A drug may be targeted to the infected cell bymeans of an anti-CtLL antibody covalently attached to a drug, whichwould bind to CtLL expressed on the surface of the cell (e.g.,macrophage). The CtLL-bound antibody conjugate would subsequently beinternalized into the infected macrophage, enhancing the bioavailabilityand efficacy of the drug. Alternatively, a pathogen that binds to a CtLLcan be targeted by linking a drug to a soluble form of the CtLL suchthat the soluble form interacts with the cognate on the pathogen thattypically interacts with a native CtLL, thus bringing the drug linked tothe soluble form in contact with the pathogen.

Macrophages or other immune cells expressing CtLL could alternativelyserve as conduits for the directed delivery of therapeutic agents todiseased cells. The therapeutic agent, or the gene encoding thetherapeutic agent, may be introduced into such immune cells expressingCtLL. The immune cells would interact specifically with diseased cellsdisplaying surface molecules recognized by CtLL, delivering the drugwhen endocytosis of the diseased target cell occurred. CtLL may also beincorporated into lipid vesicles containing a therapeutic agent. Thevesicles would interact with diseased cells or cells bearing a CtLLligand, delivering the drug to the desired target.

Tumor cells may be treated with agents to alter the structures of targetcell-surface carbohydrates or lipids to enhance the specificity oftarget cell/macrophage recognition via CtLL. This is particularlyadvantageous if CtLL recognizes “unusual” sugars or recognizes oxidizedlipids which are not normally present in the host, but can be producedin tumor cells by the administration of drugs that alter the synthesisof surface carbohydrates or oxidize the lipids on the cells surface inthe rapidly-growing tumor cells.

CtLL-specific antibodies are useful for the diagnosis of conditions anddiseases associated with expression of CtLL. The CtLL polypeptides,fragments, variants, fusion polypeptides, and the like, as set forthabove, can be employed as “immunogens” in producing antibodiesimmunoreactive therewith. Such antibodies specifically bind to thepolypeptides via the antigen-binding sites of the antibody. Specificallybinding antibodies are those that will specifically recognize and bindwith C-type lectin family polypeptides, homologues, and variants, butnot with other molecules. In one embodiment, the antibodies are specificfor polypeptides having a CtLL amino acid sequence of the invention anddo not cross-react with other polypeptides including other C-typelectin.

More specifically, the polypeptides, fragment, variants, fusionpolypeptides, and the like contain antigenic determinants or epitopesthat elicit the formation of antibodies. These antigenic determinants orepitopes can be either linear or conformational (discontinuous). Linearepitopes are composed of a single section of amino acids of thepolypeptide, while conformational or discontinuous epitopes are composedof amino acids sections from different regions of the polypeptide chainthat are brought into close proximity upon polypeptide folding. Epitopescan be identified by any of the methods known in the art. Additionally,epitopes from the polypeptides of the invention can be used as researchreagents, in assays, and to purify specific binding antibodies fromsubstances such as polyclonal sera or supernatants from culturedhybridomas. Such epitopes or variants thereof can be produced usingtechniques known in the art such as solid-phase synthesis, chemical orenzymatic cleavage of a polypeptide, or using recombinant DNAtechnology.

Both polyclonal and monoclonal antibodies to the polypeptides of theinvention can be prepared by conventional techniques. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988); Kohler andMilstein, (U.S. Pat. No. 4,376,110); the human B-cell hybridomatechnique (Kosbor et al., Immunology Today 4:72, 1983; Cole et al.,Proc. Natl. Acad. Sci. USA 80:2026, 1983); and the EBV-hybridomatechnique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy,Alan R. Liss, Inc., pp. 77-96). Hybridoma cell lines that producemonoclonal antibodies specific for the polypeptides of the invention arealso contemplated herein. Such hybridomas can be produced and identifiedby conventional techniques. For the production of antibodies, varioushost animals may be immunized by injection with a CtLL polypeptide,fragment, variant, or mutants thereof. Such host animals may include,but are not limited to, rabbits, mice, and rats, to name a few. Variousadjutants may be used to increase the immunological response. Dependingon the host species, such adjutants include, but are not limited to,Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjutants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. The monoclonalantibodies can be recovered by conventional techniques. Such monoclonalantibodies may be of any immunoglobulin class including IgG, IgM, IgE,IgA, IgD, and any subclass thereof.

In addition, techniques developed for the production of “chimericantibodies” (Takeda et al., Nature, 314:452, 1985) by splicing the genesfrom a mouse antibody molecule of appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological activity can be used. A chimeric antibody is a molecule inwhich different portions are derived from different animal species, suchas those having a variable region derived from a porcine mAb and a humanimmunoglobulin constant region. The monoclonal antibodies of theinvention also include humanized versions of murine monoclonalantibodies. Such humanized antibodies can be prepared by knowntechniques and offer the advantage of reduced immunogenicity when theantibodies are administered to humans. Procedures for the production ofchimeric and further engineered monoclonal antibodies include thosedescribed in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS84:3439, 1987), Larrick et al. (Bio/Technology 7:934, 1989), and Winterand Harris (TIPS 14:139, Can, 1993). Procedures to generate antibodiestransgenically can be found in GB 2,272,440, U.S. Pat. Nos. 5,569,825and 5,545,806 and related patents claiming priority therefrom, all ofwhich are incorporated by reference herein. When antibodies are used inhumans the antibodies are human or humanized; techniques for creatingsuch human antibodies are also known. Transgenic animals for makinghuman antibodies are available from, for example, Medarex Inc.(Princeton, N.J.) and Abgenix Inc. (Fremont, Calif.).

Antibody fragments, which recognize specific epitopes, may be generatedby known techniques. For example, such fragments include but are notlimited to: the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the (ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,Science, 246:1275, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. Techniquesdescribed for the production of single chain antibodies (U.S. Pat. No.4,946,778; Bird, Science 242:423, 1988; Huston et al., Proc. Natl. Acad.Sci. USA 85:5879, 1988; and Ward et al., Nature 334:544, 1989) can alsobe adapted to produce single chain antibodies against polypeptidescontaining CtLL amino acid sequences. In addition, antibodies to a CtLLpolypeptide can, in turn, be utilized to generate anti-idiotypeantibodies that “mimic” a CtLL polypeptide and that may bind to a CtLLpolypeptide using techniques known to those skilled in the art. (See,e.g., Greenspan & Bona, FASEB J 7(5):437, 1993; and Nissinoff, J.Immunol. 147(8):2429, 1991).

Screening procedures to identify such antibodies are known, and caninvolve immunoaffinity chromatography, for example. Antibodies can bescreened for agonistic (i.e., ligand-mimicking) properties. Suchantibodies, upon binding to a CtLL polypeptide on the cell surface, caninduce biological effects (e.g., transduction of biological signalsresulting in activation of a phosphatase) similar to the biologicaleffects induced when the naturally occurring CtLL binding partner bindsto the polypeptide on the cell surface. Agonistic antibodies can be usedto induce CtLL mediated co-stimulatory pathways or intercellularcommunication.

In addition, antibodies that block binding of a polypeptide having aCtLL sequence of the invention to its binding partner can be used toinhibit CtLL mediated intercellular communication or co-stimulation thatresults from such binding and/or to identify carbohydrate or lipidcognates of CtLL. Such blocking antibodies can be identified using anysuitable assay procedure, such as by testing antibodies for the abilityto inhibit binding of a CtLL polypeptide to certain cells expressing abinding partner (e.g., an oxidized lipid and/or a carbohydratecontaining protein) to the polypeptide or by measuring agglutination inthe presence and absence of the antibody, wherein reduction orinhibition of agglutination is indicative of an antibody that blocksinteraction of CtLL with its binding partner. Alternatively, blockingantibodies can be identified in assays for the ability to inhibit abiological effect that results from binding of a CtLL polypeptide totarget cells.

Disorders caused or exacerbated (directly or indirectly) by theinteraction of CtLL with a cell surface-binding partner or a bindingpartner on a pathogen can thus be treated. A therapeutic method involvesin vivo administration of a blocking antibody to a subject in an amounteffective to inhibit CtLL binding-mediated biological activity. As usedherein, a “subject” can be any animal, preferably a mammal (e.g.,canine, feline, bovine, porcine, equine, primates, and the like), andmost preferably a human. Monoclonal antibodies are generally preferredfor use in such therapeutic methods. In one embodiment, anantigen-binding antibody fragment is employed. Compositions comprisingan antibody against a CtLL polypeptide, and a physiologically acceptablediluent, excipient, or carrier, are provided herein.

Also provided herein are conjugates comprising a detectable (e.g.,diagnostic) or therapeutic agent attached to the antibody. Theconjugates find use in in vitro or in vivo procedures. The antibodies ofthe invention can also be used in assays to detect the presence of thepolypeptides or fragments of the invention, either in vitro or in vivo.The antibodies also can be employed in purifying polypeptides orfragments of the invention by immunoaffinity chromatography.

In another embodiment, rational drug design is used to producestructural analogs of biologically active polypeptides of interest or ofsmall molecules with which they interact, e.g., substrates, bindingagents, inhibitors, agonists, antagonists, and the like. The methodsprovided herein can be used to fashion or identify agents which are moreactive or stable forms of the polypeptide or which enhance or interferewith the function of a polypeptide in vivo (Hodgson J, Biotechnology9:19, 1991, incorporated herein by reference). In one approach, thethree-dimensional structure of a polypeptide of the invention, a ligandor binding partner, or of a polypeptide-binding partner complex, isdetermined by x-ray crystallography, by nuclear magnetic resonance, orby computer homology modeling or, most typically, by a combination ofthese approaches. Relevant structural information is used to designanalogous molecules, to identify efficient inhibitors, or to identifysmall molecules that may bind to a polypeptide of the invention. The useof C-type lectin, OLR1, CLEC1, and/or, CLEC2 polypeptide structuralinformation, preferably CtLL structural information, in molecularmodeling software systems provides for the design of inhibitors orbinding agents useful in modulating CtLL activity. A particular methodof the invention comprises analyzing the three dimensional structure ofCtLL polypeptides for likely binding sites of substrates or ligands,synthesizing a new molecule that incorporates a predictive reactivesite, and assaying the new molecule as described further herein.Examples of algorithms, software, and methods for modeling substrates orbinding agents based upon the three-dimensional structure of a proteinare described in PCT publication WO107579A2, the disclosure of which isincorporated herein.

It is also possible to isolate a target-specific antibody, selected by afunctional assay, as described further herein, and then to solve itscrystal structure thus yielding a pharmacore upon which subsequent drugdesign can be based. It is possible to bypass polypeptidecrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

CtLL antibodies are useful for the diagnosis of conditions or diseasescharacterized by expression of CtLL or in assays to monitor patientsbeing treated with CtLL agonists or inhibitors. Diagnostic assays forCtLL include methods utilizing the antibody and a label to detect CtLLin biological samples (e.g., body fluids, cells, or tissues). Thepolypeptides and antibodies of the present invention may be used with orwithout modification. Frequently, the polypeptides and antibodies willbe labeled by joining them, either covalently or noncovalently, with areporter molecule. A wide variety of reporter molecules are knownincluding enzymatic labels, fluorescent labels, radioactive labels, andthe like. A variety of protocols for measuring CtLL, using eitherpolyclonal or monoclonal antibodies are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (MA)and fluorescent activated cell sorting (FACS). Diagnosis or prognosis isperformed by quantitating the amount of CtLL in a sample compared to acontrol or normal sample. A difference in an amount of CtLL in the testsample compared to a control is indicative of a difference in expressionof CtLL and is indicative of a disease state.

The invention provides methods for identifying agents that modulate CtLLactivity or expression. Such methods included contacting a samplecontaining a CtLL polypeptide or polynucleotide with a test agent underconditions that allow for the test agent and the polynucleotide orpolypeptide to interact, and then measuring the expression or activity,respectively, of a CtLL polypeptide in the presence or absence of thetest agent.

In one embodiment, a cell containing a CtLL polynucleotide is contactedwith a test agent under conditions such that the cell and test agent areallowed to interact. Such conditions typically include normal cellculture conditions consistent with the particular cell type beingutilized and which are known in the art. It may be desirable to allowthe test agent and cell to interact under conditions associated withincreased temperature or in the presence of regents that facilitate theuptake of the test agent by the cell. A control is treated similarly butin the absence of the test agent. Alternatively, the CtLL activity orexpression may be measured prior to contact with the test agent (e.g.,the standard or control measurement) and then again following contactwith the test agent. The treated cell is then compared to the controland a difference in the expression or activity of CtLL compared to thecontrol is indicative of an agent that modulates CtLL activity orexpression.

When CtLL expression is being measured, detecting the amount of mRNAencoding a CtLL polypeptide in the cell can be quantified by, forexample, PCR or Northern blot. Where a change in the amount of CtLLpolypeptide in the sample is being measured, detecting CtLL by use ofanti-CtLL antibodies can be used to quantify the amount of CtLLpolypeptide in the cell using known techniques.

A test agent can be any molecule typically used in the modulation ofprotein activity or expression and includes, for example, smallmolecules, chemicals, peptidomimetics, antibodies, peptides,polynucleotides (e.g., antisense or ribozyme molecules), and the like.Accordingly, agents developed by computer based drug design can betested in the laboratory using the assay and methods described herein todetermine the activity of the agent on the modulation of CtLL activityor expression. Modulation of CtLL includes an increase or decrease inactivity or expression.

A CtLL polypeptide of the invention (including fragments, variants,oligomers, and other forms) are useful in a variety of assays. Forexample, a CtLL of the invention can be used to identify bindingpartners of members of the C-type lectin family of polypeptides, whichcan also be used to modulate intercellular communication,co-stimulation, macrophage activity, or pathogen infectivity.

CtLL polypeptides and fragments thereof can be used to identify bindingpartners. For example, they can be tested for the ability to bind acandidate-binding partner in any suitable assay, such as a conventionalbinding assay. To illustrate, a CtLL polypeptide or fragment thereof(preferably a soluble portion corresponding to an extracellular ofsoluble domain of CtLL) can be labeled with a detectable molecule (e.g.,a radionuclide, a chromophore, or an enzyme that catalyzes acolorimetric or fluorometric reaction, and the like). The labeledpolypeptide is contacted with cells expressing the candidate-bindingpartner. The cells then are washed to remove unbound-labeledpolypeptide, and the presence of cell-bound label is determined by asuitable technique, chosen according to the nature of the label.

In one embodiment, a binding partner is identified by the use ofantibodies to the binding partner. The ability of anti-binding partnerantibody to inhibit the binding of CtLL polypeptides reveal the bindingpartner and, indirectly, which binding activities are antagonize. CtLLpolypeptides or fragments thereof that bind to select binding partnersare further tested for the ability to disrupt interactions with, forexample, a pathogen, and to modulate other biological activities invitro and in vivo.

In another example of a binding assay, a recombinant expression vectorcontaining the candidate binding partner cDNA is transfected intoCV1-EBNA-1 cells. The cells are incubated for 1 hour at 37° C. withvarious concentrations of, for example, a soluble CtLL polypeptide/Fcfusion polypeptide. Cells are washed and incubated with a constantsaturating concentration of a ¹²⁵I-mouse anti-human IgG. After washing,cells are released via trypsinization. The mouse anti-human IgG employedabove is directed against the Fc region of human IgG and can be obtainedfrom Jackson Immunoresearch Laboratories, Inc., West Grove, Pa. Theantibody will bind to the Fc portion of any Fc polypeptide that hasbound to the cells. Cell-bound ¹²⁵I-antibody is quantified on a PackardAutogamma counter.

Where a CtLL polypeptide binds or potentially binds to anotherpolypeptide (e.g., in a receptor-ligand interaction), the CtLLpolynucleotide can also be used in interaction trap assays (see, e.g.,Gyuris et al., Cell 75:791, 1993) to identify polynucleotides encodingthe other polypeptide with which binding occurs or to identifyinhibitors of the binding interaction. Polypeptides involved in thesebinding interactions can also be used to screen for peptide or smallmolecule inhibitors or agonists of the binding interaction.

Another type of suitable binding assay is a competitive binding assay.To illustrate, biological activity of a variant can be determined byassaying for the variant's ability to compete with the nativepolypeptide for binding to the candidate-binding partner. Competitivebinding assays can be performed by conventional methodology. Reagentsthat can be employed in competitive binding assays include aradiolabeled CtLL fragment or variant and intact cells expressing CtLL(endogenous or recombinant) on the cell surface. Instead of intactcells, one could substitute a soluble binding partner/Fc fusionpolypeptide bound to a solid phase through the interaction ofPolypeptide A or G (on the solid phase) with the Fc moiety.Chromatography columns that contain Polypeptide A and G include thoseavailable from Pharmacia Biotech, Inc., Piscataway, N.J.

The influence of CtLL polypeptides, CtLL fragments (e.g., solCtLL) andantibodies on intercellular communication, co-stimulation, pathogeninfection/progression, or immune cell activity can be assayed bycontacting a cell or a group of cells with a polynucleotide,polypeptide, agonist or antagonist, to induce, enhance, suppress, orarrest intercellular communication, co-stimulation, pathogeninfection/progression, or immune cell activity in the target cells.Identification of CtLL polypeptides, agonists or antagonists can becarried out via a variety of assays known to those skilled in the art.Included in such assays are those that evaluate the ability of a CtLLpolypeptide or solCtLL to influence intercellular communication,co-stimulation, pathogen infection/progression, or immune cell activity.Such an assay would involve, for example, the analysis of cell-cellinteractions, pathogen-cell interactions in the presence of a CtLLpolypeptide or soluble fragment thereof. In one assay, the modulation ofa phosphatase activated by the ITIM moiety can be determined as ameasure of activity. In another assay, one would determine a rate ofcell-cell interaction or cell-pathogen interaction in the presence of apolypeptide having a CtLL sequence and then determine if such binding orinteraction is altered in the presence of, e.g., a soluble CtLLsequence.

In one aspect, the invention provides a method of detecting the abilityof a test agent to affect, for example, phosphatase activity in a cellor culture. In this aspect, the method comprises: (1) contacting a groupof target cells with a test agent including a polypeptide comprising aCtLL sequence (e.g., SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 or asolCtLL), a ligand or receptor for a CtLL polypeptide, or fragmentthereof, under conditions appropriate to the particular assay beingused; (2) measuring the net rate of, for example, phosphatase activityusing methods standard in the art; and (3) observing the phosphataseactivity among a group of control cells containing a CtLL polypeptideligand or fragments thereof, in the absence of a test agent, underotherwise identical conditions as the first group of cells. The testagent can function as an effector by either activating or up-regulating,or by inhibiting or down-regulating phosphatase activity. To test thecapacity of a solCtLL polypeptide to phosphatase activity, such solublepolypeptides will be added to a coculture systems containing immunecells (e.g., macrophages, DCs, T-cells and the like). Agents thatinhibit immune cell activation, as assessed by measuring phosphataseactivity, proliferation, or cytokine secretion, can be considered CtLLagonist.

In another aspect, the invention provides a method of detecting theability of a test agent to affect, for example, the cell-cellinteraction or cell-pathogen activity of the test agent on a cell orculture. In this aspect, the method comprises: (1) contacting a group oftarget cells with a test agent including a polypeptide comprising a CtLLsequence (e.g., SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 or a solCtLLbinding moiety, as described above), a ligand or receptor for a CtLLpolypeptide, or fragment thereof, under conditions appropriate to theparticular assay being used; (2) measuring the net rate of, for example,cell-cell interaction or cell-pathogen interactions; and (3) observingthe net rate of cell-cell interaction or cell-pathogen interactionsamong a group of control cells containing a CtLL polypeptide ligand orfragments thereof, in the absence of a test agent, under otherwiseidentical conditions as the first group of cells. The comparison willprovide a difference in the net rate of, for example, cell-cell orcell-pathogen interaction indicative of an agent that modulates CtLLactivity. The test agent can function as an effector by eitheractivating or up-regulating, or by inhibiting or down-regulatingcell-cell interaction or cell-pathogen interaction. To test the capacityof a solCtLL polypeptide to inhibit cell-cell interaction, such solublepolypeptides will be added to a coculture systems that contain the cellsto be tested (and antigen, if required). Samples that inhibit T or Bcell activation, for example, as assessed by proliferation or cytokinesecretion by T or B cells, will be considered to have the capacity toblock the cell interaction.

A polypeptide of the invention may exhibit cytokine production orinhibition activity, cell proliferation (either inducing or inhibiting)activity, or cell differentiation (either inducing or inhibiting)activity. Many polypeptide factors discovered to date, including mostcytokines, have exhibited activity in one or more cell proliferationassays, and hence the assays serve as a convenient confirmation ofcytokine activity. The activity of a CtLL or solCtLL polypeptide of theinvention is evidenced by any one of a number of routine factordependent cell proliferation assays for cell lines including, withoutlimitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+),2E8, RB5, DA1, 123, T1165, HT2, CtLL2, TF-1, Mo7e and CMK. The activityof a CtLL or a solCtLL polypeptide of the invention may be measured bythe following methods:

Assays for T-cell or thymocyte proliferation include, withoutlimitation, those described in: Current Protocols in Immunology, Ed. byColigan et al., Pub. Greene Publishing Associates and Wiley-Interscience(Chapter 3, In vitro assays for Mouse Lymphocyte Function 3.1-3.19;Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.137:3494, 1986; Bertagnolli et al., J. Immunol. 145:1706, 1990;Bertagnolli et al., Cell. Immunol. 133:327, 1991; Bertagnolli, et al.,J. Immunol. 149:3778, 1992; Bowman et al., J. Immunol. 152: 1756, 1994.

Assays for cytokine production and/or proliferation of spleen cells,lymph node cells or thymocytes include, without limitation, thosedescribed in: Polyclonal T cell stimulation, Kruisbeek and Shevach, Vol1 pp. 3.12.1-3.12.14, and Measurement of mouse and human Interferon γ,Schreiber, Vol 1 pp. 6.8.1-6.8.8. In Current Protocols in Immunology.Coligan eds. John Wiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic andlymphopoietic cells include, without limitation, those described in:Measurement of Human and Murine Interleukin 2 and Interleukin 4,Bottomly et al., In Current Protocols in Immunology. Coligan eds. Vol 1pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J.Exp. Med. 173:1205, 1991; Moreau et al., Nature 336:690, 1988;Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931, 1983;Measurement of mouse and human interleukin 6, Nordan, In CurrentProtocols in Immunology. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wileyand Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. U.S.A.83:1857, 1986; Measurement of human Interleukin 11, Bennett et al., InCurrent Protocols in Immunology. Coligan eds. Vol 1 pp. 6.15.1 JohnWiley and Sons, Toronto. 1991; Measurement of mouse and humanInterleukin 9, Ciarletta et al., In Current Protocols in Immunology.Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.

Assays for T-cell clone responses to antigens (which will identify,among others, polypeptides that affect APC-T cell interactions as wellas direct T-cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described in: CurrentProtocols in Immunology, Coligan eds., Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 3, Chapter 6, Chapter 7); Weinberger etal., Proc. Natl. Acad. Sci. USA 77:6091, 1980; Weinberger et al., Eur.J. Immun. 11:405, 1981; Takai et al., J. Immunol. 137:3494, 1986; Takaiet al., J. Immunol. 140:508, 1988.

Assays for thymocyte or splenocyte cytotoxicity include, withoutlimitation, Current Protocols in Immunology, Coligan eds., Pub. GreenePublishing Associates and Wiley-Interscience (pp. 3.1-3.19; Chapter 7);Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488, 1981; Herrmann etal., J. Immunol. 128:1968, 1982; Handa et al., J. Immunol. 135:1564,1985; Takai et al., J. Immunol. 137:3494, 1986; Takai et al., J.Immunol. 140:508, 1988; Bowman et al., J. Virol. 61:1992; Bertagnolli etal., Cell. Imm. 133:327, 1991; Brown et al., J. Immunol. 153:3079, 1994.

Assays for T-cell-dependent IgG responses and isotype switching (whichwill identify, among others, polypeptides that modulate T-cell dependentantibody responses and that affect Th1/Th2 profiles) include, withoutlimitation, those described in: Maliszewski, J. Immunol. 144:3028, 1990;and Assays for B cell function: In vitro antibody production, Mond andBrunswick, In Current Protocols in Immunology. Coligan eds. Vol 1 pp.3.8.1-3.8.16, Wiley and Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, amongothers, polypeptides that generate predominantly Th1 and CTL responses)include, without limitation, those described in: Current Protocols inImmunology, Coligan eds., Pub. Greene Publishing Associates andWiley-Interscience (In vitro assays for Mouse Lymphocyte Function pp3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., 1986,supra; Takai et al., 1988, supra; Bertagnolli et al., J. Immunol.149:3778, 1992.

Dendritic cell-dependent assays (which will identify, among others,polypeptides expressed by dendritic cells that activate naive T-cells)include, without limitation, those described in: Guery et al., J.Immunol. 134:536, 1995; Inaba et al., J. of Exp. Med. 173:549, 1991;Macatonia et al., J. Immunol. 154:5071, 1995; Porgador et al., J. ofExp. Med. 182:255, 1995; Nair et al., J. Virol. 67:4062, 1993; Huang etal., Science 264:961, 1994; Macatonia et al., J. of Exp. Med. 169:1255,1989; Bhardwaj et al., J. Clin. Invest. 94:797, 1994; and Inaba et al.,J. of Exp. Med. 172:631, 1990.

Assays for lymphocyte survival/apoptosis (which will identify, amongothers, polypeptides that prevent apoptosis after superantigen inductionand polypeptides that regulate lymphocyte homeostasis) include, withoutlimitation, those described in: Darzynkiewicz et al., Cytometry 13:795,1992; Gorczyca et al., Leukemia 7:659, 1993; Gorczyca et at, CancerResearch 53:1945, 1993; Itoh et al., Cell 66:233, 1991; Zacharchuk, J.Immunol. 145:4037, 1990; Zamai et al., Cytometry 14:891, 1993; Gorczycaet al., Int. J. of Oncology 1:639, 1992.

Assays for polypeptides that influence early steps of T-cell commitmentand development include, without limitation, those described in: Anticaet al., Blood 84:111, 1994; Fine et al., Cell. Immunol. 155:111, 1994;Galy et al., Blood 85:2770, 1995; Toki et al., Proc. Nat. Acad Sci. USA88:7548, 1991.

Assays for embryonic stem cell differentiation (which will identify,among others, polypeptides that influence embryonic differentiationhematopoiesis) include, without limitation, those described in:Johansson et al. Cell. Biol. 15:141, 1995; Keller et al., Mol. and Cell.Biol. 13:473, 1993; McClanahan et al., Blood 81:2903, 1993.

Assays for stem cell survival and differentiation (which will identify,among others, polypeptides that regulate lympho-hematopoiesis) include,without limitation, those described in: Methylcellulose colony formingassays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y.1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;Primitive hematopoietic colony forming cells with high proliferativepotential, McNiece and Briddell, In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, N.Y.1994; Neben et al., Exp. Hematol. 22:353, 1994; Cobblestone area formingcell assay, Ploemacher, In Culture of Hematopoietic Cells. Freshney, etal. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long termbone marrow cultures in the presence of stromal cells, Spooncer et al.In Culture of Hematopoietic Cells. Freshney, et al. eds. Vol pp.163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term cultureinitiating cell assay, Sutherland, In Culture of Hematopoietic Cells.Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, N.Y.1994.

Assays for tissue generation activity include, without limitation, thosedescribed in: Patent Publication No. W095/16035 (bone, cartilage,tendon); Patent Publication No. WO95/05846 (nerve, neuronal); PatentPublication No. WO91/07491 (skin, endothelium). Assays for wound healingactivity include, without limitation, those described in: Winter,Epidermal Wound Healing, pps. 71-112 (Maibach, and Rovee, eds.), YearBook Medical Publishers, Inc., Chicago, as modified by Eaglstein andMertz, J. Invest. Dermatol 71:382-84 (1978).

Assays for activin/inhibin activity include, without limitation, thosedescribed in: Vale et al., Endocrinol. 91:562, 1972; Ling et al., Nature321:779, 1986; Vale et al., Nature 321:776, 1986; Mason et al., Nature318:659, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091, 1986.

Assays for cell movement and adhesion include, without limitation, thosedescribed in: Current Protocols in Immunology, Coligan eds., Pub: GreenePublishing Associates and Wiley-Interscience (Chapter 6,6.12.1-6.12.28); Taub et al. J. Clin. Invest. 95:1370, 1995; Lind et al.APMIS 103:140, 1995; Muller et al. Eur. J. Immunol. 25: 1744; Gruber etal. J. Immunol. 152:5860, 1994; Johnston et al. J. Immunol. 153:1762,1994.

Assay for hemostatic and thrombolytic activity include, withoutlimitation, those described in: Linet et al., J. Clin. Pharmacol.26:131, 1986; Burdick et al., Thrombosis Res. 45:413,1987; Humphrey etal., Fibrinolysis 5:71, 1991; Schaub, Prostaglandins 35:467, 1988.

Assays for receptor-ligand activity include, without limitation, thosedescribed in: Current Protocols in Immunology, Coligan eds., Pub. GreenePublishing Associates and Wiley-Interscience (Chapter 7.28.1-7.28.22),Takai et al., Proc. Natl. Acad. Sci. USA 84:6864, 1987; Bierer et al.,J. Exp. Med. 168:1145, 1988; Rosenstein et al., J. Exp. Med. 169:149,1989; Stoltenborg et al., J. Immunol. Methods 175:59, 1994; Stitt etal., Cell 80:661, 1995.

Assays for cadherin adhesive and invasive suppressor activity include,without limitation, those described in: Hortsch et al. J Biol. Chem.270(32):18809, 1995; Miyaki et al. Oncogene 11: 2547, 1995; Ozawa et al.Cell 63:1033, 1990.

A polynucleotide encoding a polypeptide comprising a CtLL sequenceprovided by the invention can be used for numerous diagnostic or otheruseful purposes. A polynucleotide of the invention (e.g., SEQ ID NO:1,3, 5, 7, 9, 11, 13, or 15) can be used as markers for tissues in whichthe corresponding polypeptide is preferentially expressed, as molecularweight markers on Southern gels, as chromosome markers or tags toidentify chromosomes or to map related gene positions, to compare withendogenous DNA sequences in subjects to identify potential geneticdisorders, as probes to hybridize and thus discover novel relatedpolynucleotides, as a source of information to derive PCR primers forgenetic fingerprinting, as a probe to “subtract-out” knownpolynucleotides in the process of discovering other novel nucleic acids,as an antigen to raise anti-DNA antibodies or elicit another immuneresponse, and for gene therapy.

Probes and Primers. Among the uses of the disclosed CtLLpolynucleotides, and combinations of fragments thereof, is the use offragments as probes or primers. Such fragments generally comprise atleast about 17 contiguous nucleotides of a DNA sequence. In otherembodiments, a DNA fragment comprises at least 30, or at least 60contiguous nucleotides of a DNA sequence. The basic parameters affectingthe choice of hybridization conditions and guidance for devisingsuitable conditions are set forth by Sambrook et al., 1989 and aredescribed in detail above. Using knowledge of the genetic code incombination with the amino acid sequences set forth above, sets ofdegenerate oligonucleotides can be prepared. Such oligonucleotides areuseful as primers, e.g., in polymerase chain reactions (PCR), wherebyDNA fragments are isolated and amplified. In certain embodiments,degenerate primers can be used as probes for non-human geneticlibraries. Such libraries would include but are not limited to cDNAlibraries, genomic libraries, and even electronic EST (express sequencetag) or DNA libraries. Homologous sequences identified by this methodwould then be used as probes to identify non-human homologues of theCtLL sequence identified herein.

Chromosome Mapping. The polynucleotides encoding CtLL polypeptides, andthe disclosed fragments and combinations of these polynucleotides, canbe used by those skilled in the art using known techniques to identifythe human chromosome to which these sequences map. Useful techniquesinclude, but are not limited to, using the sequence or portions,including oligonucleotides, as a probe in various known techniques suchas radiation hybrid mapping (high resolution), in situ hybridization tochromosome spreads (moderate resolution), and Southern blothybridization to hybrid cell lines containing individual humanchromosomes (low resolution). Information about radiation hybrid mappingcan be found on the worldwideweb (www) at:genome.wi.mit.edu/ftp/distribution/human_STS_releases/july97/07-97.INTRO.html.

A polynucleotide encoding a polypeptide comprising a CtLL sequence ofthe invention, and the disclosed fragments and combinations of thesepolynucleotides can be used to analyze abnormalities associated with thegenes corresponding to CtLL polypeptides. This enables one todistinguish conditions in which this marker is rearranged or deleted. Inaddition, polynucleotides of the invention or a fragment thereof can beused as a positional marker to map other genes of unknown location. Thepolynucleotide can be used in developing treatments for any disordermediated (directly or indirectly) by defective, or insufficient amountsof, genes corresponding to the polynucleotides of the invention. Thepolynucleotides and associated sequences disclosed herein permit thedetection of defective genes, and the replacement thereof with normalgenes. Defective genes can be detected in in vitro diagnostic assays,and by comparison of the polynucleotide sequences disclosed herein withthat of a gene derived from a subject suspected of harboring a defect inthis gene or having a CtLL-associated disorder.

Uses of CtLL polypeptides and peptide fragments thereof include, but arenot limited to, the following: delivery agents; therapeutic and researchreagents; molecular weight and isoelectric focusing markers; controlsfor peptide fragmentation; identification of unknown polypeptides; andpreparation of antibodies.

The CtLL polypeptides (e.g., SEQ ID NO:2, 4, 6, 8, 10, 12, 14, and 16,and fragments, including solCtLL polypeptides thereof) of the inventioncan be used as purification reagents. For example, CtLL or solCtLLpolypeptides can be attached to a solid support material and used topurify its binding partners by affinity chromatography. In particularembodiments, a polypeptide is attached to a solid support byconventional procedures. As one example, chromatography columnscontaining functional groups that will react with amino acid side chainsof polypeptides are available (Pharmacia Biotech, Inc., Piscataway,N.J.). In an alternative embodiment, a solCtLL-Fc polypeptide isattached to Polypeptide A- or G-containing chromatography columnsthrough interaction with the Fc moiety. The polypeptide also finds usein purifying or identifying cells that express a binding partner on thecell surface. Polypeptides are bound to a solid phase such as a columnchromatography matrix or a similar suitable substrate. For example,magnetic microspheres can be coated with the polypeptides and held in anincubation vessel through a magnetic field. Suspensions of cell mixturescontaining the binding partner expressing cells are contacted with thesolid phase having the polypeptides thereon. Cells expressing thebinding partner on the cell surface bind to the polypeptides on thesolid phase, and unbound cells then are washed away. Alternatively, thepolypeptides can be conjugated to a detectable moiety, then incubatedwith cells to be tested for binding partner expression. Afterincubation, unbound-labeled matter is removed and the presence orabsence of the detectable moiety on the cells is determined.

Carriers and Delivery Agents. The polypeptides also find use as carriersfor delivering agents attached thereto to cells or pathogens bearingidentified binding partners. Thus, solCtLL polypeptides can be used todeliver diagnostic or therapeutic agents to such cells or pathogens inin vitro or in vivo procedures. Detectable (diagnostic) and therapeuticagents that can be attached to a CtLL or solCtLL polypeptide include,but are not limited to, toxins, cytotoxic agents, drugs, radionuclides,chromophores, enzymes that catalyze a colorimetric or fluorometricreaction, and the like, with the particular agent being chosen accordingto the intended application. Among the toxins are ricin, abrin,diphtheria toxin, Pseudomonas aeruginosa exotoxin A, ribosomalinactivating polypeptides, mycotoxins such as trichothecenes, andderivatives and fragments (e.g., single chains) thereof. Radionuclidessuitable for diagnostic use include, but are not limited to, ¹²³I, ¹³¹I,^(99m)Tc, ¹¹¹In, and ⁷⁶Br. Examples of radionuclides suitable fortherapeutic use are ¹³¹I, ²¹¹At, ⁷⁷Br, ¹⁸⁶Re, ¹⁸⁸Re, ²¹²Pb, ²¹²Bi,¹⁰⁹Pd, ⁶⁴Cu, and ⁶⁷Cu. Such agents can be attached to the polypeptide byany suitable conventional procedure. A CtLL or solCtLL polypeptidecomprises functional groups on amino acid side chains that can bereacted with functional groups on a desired agent to form covalentbonds, for example. Alternatively, the polypeptide or agent can bederivatized to generate or attach a desired reactive functional group.The derivatization can involve attachment of one of the bifunctionalcoupling reagents available for attaching various molecules topolypeptides (Pierce Chemical Company, Rockford, Ill.). Of particularinterest are soluble CtLL domains that can be used to target cellsexpressing a binding partner for a CtLL polypeptide. Such soluble CtLLpolypeptides can be used to target reagents to cells or pathogensexpressing, for example, a cognate to a CtLL polypeptide. Similarly, andas discussed more fully below, antibodies specific for a CtLLpolypeptide can be labeled with a diagnostic or therapeutic agent andused to target the diagnostic or therapeutic to cells expressing a CtLLpolypeptide.

CtLL polypeptides and fragments (e.g., solCtLL and fragments) can beemployed in modulating a biological activity of a C-type lectinpolypeptide, particularly CtLL polypeptide, in in vitro or in vivoprocedures. Encompassed within the invention are domains of CtLLpolypeptides that act as modulators of native CtLL polypeptide functionwhen expressed as fragments or as components of fusion polypeptides. Forexample, a substantially purified polypeptide domain of the inventioncan be used to inhibit binding of a CtLL polypeptide to endogenousbinding partners. Such use effectively would block CtLL interactions andinhibit CtLL activities. In still another aspect of the invention, asoluble form of a CtLL binding partner is used to bind to, andcompetitively inhibit activation of the endogenous CtLL polypeptide.

Antibodies that bind to CtLL polypeptides can inhibit CtLL polypeptideactivity and may act as antagonists. For example, antibodies thatspecifically bind to one or more epitopes of a CtLL polypeptide, orepitope of conserved variants of CtLL polypeptides, or fragments can beused to inhibit CtLL activity. By “specifically bind” means that anantibody to a CtLL polypeptide or fragment thereof will not cross-reactwith unrelated polypeptides. Preferably such an antibody will notcross-react with other members of the C-type lectin family of proteins.

In an alternative aspect, the invention further encompasses the use ofagonists of CtLL activity to treat or ameliorate the symptoms of adisease for which increased C-type lectin activity is beneficial. In apreferred aspect, the invention entails administering compositionscomprising a CtLL polynucleotide (e.g., comprising SEQ ID NO:1, 3, 5, 7,9, 11, 13, and/or 15) or fragment thereof or a polypeptide comprising aCtLL amino acid sequence (e.g., SEQ ID NO:2, 4, 6, 8, 10, 12, 14, and/or16) or fragment thereof. The administering may be to cells in vitro, tocells ex vivo, to cells in vivo, and/or to a multicellular organism. Onetherapeutic form includes soluble forms of a CtLL polypeptide. Such asoluble CtLL polypeptide will bind to its binding partner (e.g., anoxidized lipid and/or a carbohydrate-containing moiety) and stimulate abiological activity associated with the binding partner.

In still another aspect of the invention, the compositions compriseadministering a polynucleotide encoding a CtLL or a solCtLL polypeptidefor expression in a host organism for treatment of disease. Particularlypreferred in this regard is expression in a human subject for treatmentof a dysfunction associated with aberrant (e.g., decreased or increased)endogenous activity of a CtLL polypeptide. Furthermore, the inventionencompasses the administration of compounds found to increase theendogenous activity of polypeptides comprising a CtLL amino acidsequence to cells and/or organisms. One example of compounds thatincrease CtLL polypeptide activity are antibodies, preferably monoclonalantibodies, that bind to CtLL polypeptides and increase or stimulateCtLL polypeptide activity by causing constitutive intracellularsignaling (or “ligand mimicking”), or by preventing the binding of anative inhibitor of CtLL polypeptide activity.

Due to the multiplicity and interconnectedness of biological pathwaysand interactions, a CtLL polypeptide, solCtLL polypeptide, fragments ofthe foregoing, variant, antagonist, agonist, antibody, and bindingpartner of the invention can be useful for treating medical conditionsand diseases associated with cell-cell, cell-pathogen interactions,atherosclerosis, coronary diseases and disorders, cell proliferativediseases and disorders, and inflammatory disease and disorder asdescribed further herein. The therapeutic molecule or molecules to beused will depend on the etiology of the condition to be treated and thebiological pathways involved, and will consider that different variants,antagonists, and binding partners of CtLL polypeptides may have similaror different effects. Accordingly, a CtLL agonist or antagonist maymodulate immune cell activity and inflammation, such as release ofgrowth factors, adhesion proteins, and inflammatory factors.

The disclosed CtLL polypeptides, solCtLL polypeptides, fragmentsthereof, antibodies, compositions and combination therapies describedherein are useful in medicines for treating bacterial; viral orprotozoal infections, and complications resulting therefrom.Cardiovascular disorders are treatable with the disclosed CtLLpolypeptides, solCtLL polypeptides, fragments thereof, antibodies,compositions and combination therapies, including aortic aneurysms;arteritis; vascular occlusion; complications of coronary by-passsurgery; ischemia/reperfusion injury; heart disease; heart failure; andmyocardial infarction. In addition, the CtLL polypeptides, solCtLLpolypeptides, fragments thereof, antibodies, compositions andcombination therapies of the invention can be used to treat chronic painconditions, to treat various disorders of the endocrine system,conditions of the gastrointestinal system, disorders of thegenitourinary system, and anemias and hematological disorders.

In addition, the polypeptides, fragments thereof, antibodies,compositions, and combination therapies of the invention haveapplication to cell proliferative disorders, including cancer and cancercell metastasis. Also provided herein are methods for using CtLLpolypeptides, solCtLL polypeptides, fragments thereof, antibodies,compositions and combination therapies to treat various hematologic andoncologic disorders. For example, soluble CtLL domains can be used totreat various forms of cancer, including acute myelogenous leukemia,Epstein-Barr virus-positive nasopharyngeal carcinoma, glioma, colon,stomach, prostate, renal cell, cervical and ovarian cancers, lung cancer(SCLC and NSCLC), including cancer-associated cachexia, fatigue,asthenia, paraneoplastic syndrome of cachexia, and hypercalcemia bymodulating lectin-associated interactions.

Additional diseases treatable with the polypeptides, fragments,antibodies, compositions or combination therapies of the invention aresolid tumors, including sarcoma, osteosarcoma, and carcinoma, such asadenocarcinoma (e.g., breast cancer) and squamous cell carcinoma.Administration of a solCtLL domain can modulate cell-cell andcell-matrix interactions of such tumor cells and/or modulate theangiogenesis and blood supply to such tumors.

In addition, the CtLL polypeptides, solCtLL polypeptides, fragmentsthereof, antibodies, compositions and combination therapies are usefulfor treating leukemia, including acute myelogenous leukemia, chronic oracute lymphoblastic leukemia and hairy cell leukemia. Other malignancieswith invasive metastatic potential that can be treated with the CtLLpolypeptides, solCtLL polypeptides, fragments thereof, antibodies,compositions and combination therapies, include multiple myeloma,various lymphoproliferative disorders such as autoimmunelymphoproliferative syndrome (ALPS), chronic lymphoblastic leukemia,hairy cell leukemia, chronic lymphatic leukemia, peripheral T-celllymphoma, small lymphocytic lymphoma, mantle cell lymphoma, follicularlymphoma, Burkitt's lymphoma, Epstein-Barr virus-positive T celllymphoma, histiocytic lymphoma, Hodgkin's disease, diffuse aggressivelymphoma, acute lymphatic leukemias, T gamma lymphoproliferativedisease, cutaneous B cell lymphoma, cutaneous T cell lymphoma (i.e.,mycosis fungoides), and Sézary syndrome.

A combination of at least one CtLL polypeptide, solCtLL polypeptides,fragment thereof, or antibody, and one or more anti-angiogenesis factorsor other therapeutic agent(s) may be administered to the subject. Theadditional therapeutic agent(s) may be administered prior to,concurrently with, or following the administration of the CtLLpolypeptide, solCtLL polypeptides, fragment thereof, or antibody. Theuse of more than one therapeutic agent is particularly advantageous whenthe subject that is being treated has a solid tumor. In some embodimentsof the invention, the treatment further comprises treating the mammalwith radiation. Radiation, including brachytherapy and teletherapy, maybe administered prior to, concurrently with, or following theadministration of the CtLL polypeptide, solCtLL polypeptides, fragment,antibody, or CtLL binding partner and/or additional therapeuticagent(s).

In some embodiments the method includes the administration of, inaddition to CtLL polypeptide, solCtLL polypeptides, fragment thereof, orantibody, one or more therapeutics selected from the group consisting ofalkylating agents, antimetabolites, vinca alkaloids and otherplant-derived chemotherapeutics, antitumor antibiotics, antitumorenzymes, topoisomerase inhibitors, platinum analogs, adrenocorticalsuppressants, hormones and antihormones, antibodies, immunotherapeutics,radiotherapeutics, and biological response modifiers.

In some embodiments the method includes, administration of, in additionto a CtLL polypeptide, solCtLL polypeptides, fragment thereof, orantibody, one or more therapeutics selected from the group consisting ofcisplatin, cyclophosphamide, mechloretamine, melphalan, bleomycin,carboplatin, fluorouracil, 5-fluorodeoxyuridine, methotrexate, taxol,asparaginase, vincristine, and vinblastine, lymphokines and cytokinessuch as interleukins, interferons (α, β or δ) and TNF, chlorambucil,busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine,cytarabine, mercaptopurine, thioguanine, vindesine, etoposide,teniposide, dactinomycin, daunorubicin, doxorubicin, bleomycin,plicamycin, mitomycin, L-asparaginase, hydroxyurea, methylhydrazine,mitotane, tamoxifen, fluoxymesterone, IL-8 inhibitors, angiostatin,endostatin, kringle 5, angiopoietin-2 or other antagonists ofangiopoietin-1, antagonists of platelet-activating factor, antagonistsof basic fibroblast growth factor, and COX-2 inhibitors.

In some embodiments the method includes administration of, in additionto a CtLL polypeptide, solCtLL polypeptides, fragment thereof, orantibody, one or more therapeutic polypeptides, including soluble formsthereof, selected from the group consisting of Flt3 ligand (see, U.S.Pat. No. 5,554,512), CD40 ligand (see, U.S. Pat. No. 5,716,805), IL-2,IL-12, 4-1BB ligand (see, U.S. Pat. No. 5,674,704), anti-4-1BBantibodies, TRAIL, TNF antagonists and TNF receptor antagonistsincluding TNFR/Fc, Tek antagonists, TWEAK antagonists and TWEAK-R (see,U.S. Ser. Nos. 60/172,878 and 60/203,347 and Feng et al., Am. J. Pathol.156(4):1253) antagonists including TWEAK-R/Fc, VEGF antagonistsincluding anti-VEGF antibodies, VEGF receptor (including VEGF-R1 andVEGF-R2, also known as Flt1 and Flk1 or KDR) antagonists, CD148 (alsoreferred to as DEP-1, ECRTP, and PTPRJ, see Takahashi et al., J. Am.Soc. Nephrol. 10:2135-45, 1999; and PCT Publication No. WO 00/15258, 23Mar. 2000) binding proteins, and nectin-3 (see, Satoh-Horikawa et al.,J. Biol. Chem. 275(14):10291, 2000; GenBank accession numbers of humannectin-3 nucleic acid and polypeptide sequences are AF282874 andAAF97597, respectively) antagonists.

In some preferred embodiments a CtLL polypeptide, solCtLL polypeptides,fragment thereof, or antibody of the invention is used as a componentof, or in combination with, “metronomic therapy,” such as that describedby Browder et al. and Klement et al. (Cancer Research 60:1878, 2000; J.Clin. Invest. 105(8):R15, 2000; see also Barinaga, Science 288:245,2000).

This invention provides compounds, compositions, and methods fortreating a subject, preferably a mammalian subject, and most preferablya human subject, who is suffering from a CtLL-associated disorder. SuchCtLL-associated disorders include conditions caused (directly orindirectly) or exacerbated by binding between a polypeptide having anCtLL sequence (e.g., SEQ ID NO:2, 4, 6, 8, 10, 12, 14, or 16) and itsbinding partner (e.g., an oxidized lipid and/or a carbohydrate moiety).For purposes of this disclosure, the terms “illness,” “disease,”“disorder,” “medical condition,” “abnormal condition” and the like areused interchangeably with the term “medical disorder.” The terms“treat”, “treating”, and “treatment” used herein include curative,preventative (e.g., prophylactic) and palliative or ameliorativetreatment. For such therapeutic uses, CtLL polypeptides and fragments(including solCtLL and fragments thereof), CtLL polynucleotides encodinga CtLL polypeptide or fragment, and/or agonists or antagonists of theCtLL polypeptide such as antibodies or solCtLL can be administered tothe subject in need through known means. Compositions of the inventioncan contain a polypeptide in any form described herein, such as nativepolypeptides, variants, derivatives, oligomers, and biologically activefragments. In particular embodiments, the composition comprises asoluble polypeptide or an oligomer comprising soluble CtLL (solCtLL)polypeptides.

In practicing the method of treatment or use of the invention, atherapeutically effective amount of a therapeutic agent of the inventionis administered to a subject having a condition to be treated,preferably to treat or ameliorate diseases associated with the activityof a CtLL polypeptide. “Therapeutic agent” includes without limitationany CtLL polypeptide, solCtLL polypeptides, fragment, and variant;polynucleotide encoding a CtLL polypeptide, solCtLL polypeptide,fragment, and variant; agonists or antagonists of the a CtLL polypeptidesuch as antibodies; a CtLL polypeptide binding partner; complexes formedfrom a CtLL polypeptide, solCtLL polypeptide, fragment, variant, andbinding partner, and the like. As used herein, the term “therapeuticallyeffective amount” means the total amount of each therapeutic agent orother active component of the pharmaceutical composition or method thatis sufficient to show a meaningful subject benefit, e.g., treatment,healing, prevention or amelioration of the relevant medical condition,or an increase in rate of treatment, healing, prevention or ameliorationof such conditions. When applied to an individual therapeutic agent oractive ingredient, administered alone, the term refers to thatingredient alone. When applied to a combination, the term refers tocombined amounts of the ingredients that result in the therapeuticeffect, whether administered in combination, serially or simultaneously.As used herein, the phrase “administering a therapeutically effectiveamount” of a therapeutic agent means that the subject is treated withsaid therapeutic agent in an amount and for a time sufficient to inducean improvement, and preferably a sustained improvement, in at least oneindicator that reflects the severity of the disorder. An improvement isconsidered “sustained” if the subject exhibits the improvement on atleast two occasions separated by one or more weeks. The degree ofimprovement is determined based on signs or symptoms, and determinationsmay also employ questionnaires that are administered to the subject,such as quality-of-life questionnaires. Various indicators that reflectthe extent of the subject's illness may be assessed for determiningwhether the amount and time of the treatment is sufficient. The baselinevalue for the chosen indicator or indicators is established byexamination of the subject prior to administration of the first dose ofthe therapeutic agent. Preferably, the baseline examination is donewithin about 60 days of administering the first dose. If the therapeuticagent is being administered to treat acute symptoms, the first dose isadministered as soon as practically possible after the injury hasoccurred. Improvement is induced by administering therapeutic agentssuch as a CtLL polypeptide, solCtLL polypeptide, fragment, antibody, orCtLL binding partner until the subject manifests an improvement overbaseline for the chosen indicator or indicators. In treating chronicconditions, this degree of improvement is obtained by repeatedlyadministering this medicament over a period of at least a month or more,e.g., for one, two, or three months or longer, or indefinitely. A periodof one to six weeks, or even a single dose, often is sufficient fortreating acute conditions. Although the extent of the subject's illnessafter treatment may appear improved according to one or more indicators,treatment may be continued indefinitely at the same level or at areduced dose or frequency. Once treatment has been reduced ordiscontinued, it later may be resumed at the original level if symptomsshould reappear.

One skilled in the art will recognize that suitable dosages will vary,depending upon such factors as the nature and severity of the disorderto be treated, the subject's body weight, age, general condition, andprior illnesses and/or treatments, and the route of administration.Preliminary doses can be determined according to animal tests, and thescaling of dosages for human administration is performed according toart-accepted practices such as standard dosing trials. For example, thetherapeutically effective dose can be estimated initially from cellculture assays. The dosage will depend on the specific activity of thecompound and can be readily determined by routine experimentation. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture, while minimizing tonicities. Suchinformation can be used to more accurately determine useful doses inhumans. Ultimately, the attending physician will decide the amount ofpolypeptide of the invention with which to treat each individualsubject. Initially, the attending physician will administer low doses ofpolypeptide of the invention and observe the subject's response. Largerdoses of polypeptide of the invention may be administered until theoptimal therapeutic effect is obtained for the subject, and at thatpoint the dosage is not increased further. It is contemplated that thevarious pharmaceutical compositions used to practice the method of theinvention should contain about 0.01 ng to about 100 mg (preferably about0.1 ng to about 10 mg, more preferably about 0.1 microgram to about 1mg) of a polypeptide of the invention per kg body weight. In oneembodiment of the invention, a CtLL polypeptide, solCtLL polypeptide,fragment, antibody, or CtLL binding partner is administered one time perweek to treat the various medical disorders disclosed herein. In anotherembodiment polypeptide, fragment, antibody, or CtLL binding partner isadministered at least two times per week and in another embodiment atleast three times per week. If injected, the effective amount of a CtLLpolypeptide, solCtLL polypeptide, fragment, antibody, or CtLL bindingpartner per adult dose ranges from 1-20 mg/m², and preferably is about5-12 mg/m². Alternatively, a flat dose may be administered whose amountmay range from 5-100 mg/dose. Exemplary dose ranges for a flat dose tobe administered by subcutaneous injection are 5-25 mg/dose, 25-50mg/dose and 50-100 mg/dose. In one embodiment of the invention, thevarious indications described herein are treated by administering apreparation acceptable for injection containing a CtLL polypeptide,solCtLL polypeptide, fragment, antibody, or CtLL binding partner at 25mg/dose, or alternatively, containing 50 mg per dose. The 25 mg or 50 mgdose may be administered repeatedly, particularly for chronicconditions. If a route of administration other than injection is used,the dose is appropriately adjusted in accord with standard medicalpractices.

In many instances, an improvement in a subject's condition will beobtained by injecting a dose of about 25 mg of a CtLL polypeptide,solCtLL polypeptide, fragment, antibody, or CtLL binding partner one tothree times per week over a period of at least three weeks, or a dose of50 mg of a CtLL polypeptide, fragment, antibody, or CtLL binding partnerone or two times per week for at least three weeks (a treatment forlonger periods may be necessary to induce the desired degree ofimprovement). For incurable chronic conditions, the regimen may becontinued indefinitely, with adjustments being made to dose andfrequency if such are deemed necessary by the subject's physician. Theforegoing doses are examples for an adult subject who is a person who is18 years of age or older. For pediatric subjects (age 4-17), a suitableregimen involves the subcutaneous injection of 0.4 mg/kg, up to amaximum dose of 25 mg of a CtLL polypeptide, solCtLL polypeptide,fragment, antibody, or CtLL binding partner, administered bysubcutaneous injection one or more times per week. If an antibodyagainst a CtLL polypeptide is used as a CtLL polypeptide antagonist, apreferred dose range is 0.1 to 20 mg/kg, and more preferably is 1-10mg/kg. Another preferred dose range for an anti-CtLL polypeptideantibody is 0.75 to 7.5 mg/kg of body weight. Humanized antibodies arepreferred. Such antibodies may be injected or administeredintravenously.

Compositions comprising an effective amount of a CtLL polypeptides,solCtLL polypeptides, fragments thereof, and antibodies of the invention(from whatever source derived, including without limitation fromrecombinant and non-recombinant sources), in combination with othercomponents such as a physiologically acceptable diluent, carrier, orexcipient, are provided herein. The term “pharmaceutically acceptable”means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).Formulations suitable for administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation isotonic with theblood of the recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents or thickening agents. Thepolypeptides can be formulated according to known methods used toprepare pharmaceutically useful compositions. They can be combined inadmixture, either as the sole active material or with other known activematerials suitable for a given indication, with pharmaceuticallyacceptable diluents (e.g., saline, Tris-HCl, acetate, and phosphatebuffered solutions), preservatives (e.g., thimerosal, benzyl alcohol,parabens), emulsifiers, solubilizers, adjuvants and/or carriers.Suitable formulations for pharmaceutical compositions include thosedescribed in Remington's Pharmaceutical Sciences, 16th ed. 1980, MackPublishing Company, Easton, Pa. In some embodiments the polypeptide mayundergo pegylation to assist in adsorption or uptake. For example, suchcompositions can be complexed with polyethylene glycol (PEG), metalions, or incorporated into polymeric compounds such as polyacetic acid,polyglycolic acid, hydrogels, dextran, and the like, or incorporatedinto liposomes, microemulsions, micelles, unilamellar or multilamellarvesicles, erythrocyte ghosts or spheroblasts. Suitable lipids forliposomal formulation include, without limitation, monoglycerides,diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bileacids, and the like. Preparation of such liposomal formulations iswithin the level of skill in the art, as disclosed, for example, in U.S.Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028;and U.S. Pat. No. 4,737,323, all of which are incorporated herein byreference. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance, and are thus chosen according to the intended application, sothat the characteristics of the carrier will depend on the selectedroute of administration. In one preferred embodiment of the invention,sustained-release forms of a CtLL polypeptides, solCtLL polypeptides,fragments thereof, and/or antibodies are used. Examples ofsustained-release forms suitable for use in the disclosed methodsinclude, but are not limited to, a CtLL polypeptide that is encapsulatedin a slowly-dissolving biocompatible polymer (such as the alginatemicroparticles described in U.S. Pat. No. 6,036,978), admixed with sucha polymer (including topically applied hydrogels), and or encased in abiocompatible semi-permeable implant.

A CtLL polypeptide or solCtLL polypeptide of the invention may be activein multimers (e.g., heterodimers or homodimers) or complexes with itselfor other polypeptides. As a result, pharmaceutical compositions of theinvention may comprise a polypeptide of the invention in such multimericor complexed form. The pharmaceutical composition of the invention maybe in the form of a complex of the polypeptide(s) of invention. Theinvention further includes the administration of a CtLL polypeptide,solCtLL polypeptide, fragment, antibody, or CtLL binding partnerconcurrently with one or more other drugs that are administered to thesame subject in combination, each drug being administered according to aregimen suitable for that medicament. “Concurrent administration”encompasses simultaneous or sequential treatment with the components ofthe combination, as well as regimens in which the drugs are alternated,or wherein one component is administered long-term and the other(s) areadministered intermittently. Components may be administered in the sameor in separate compositions, and by the same or different routes ofadministration. Examples of components that may be included in thepharmaceutical composition of the invention are cytokines, lymphokines,or other hematopoietic factors such as: M-CSF, GM-CSF, TNF, IL-1, IL-2,IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-17, IL-18, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF,thrombopoietin, stem cell factor, and erythropoietin. The pharmaceuticalcomposition may further contain other agents that either enhance theactivity of the polypeptide or compliment its activity or use intreatment. Such additional factors and/or agents may be included in thepharmaceutical composition to produce a synergistic effect with apolypeptide of the invention, or to minimize side effects. Conversely, aCtLL polypeptide, a solCtLL polypeptide, fragment, antibody, or CtLLbinding partner of the invention may be included in formulations with aparticular cytokine, lymphokine, other hematopoietic factor,thrombolytic or anti-thrombotic factor, or anti-inflammatory agent tominimize side effects of the cytokine, lymphokine, other hematopoieticfactor, thrombolytic or anti-thrombotic factor, or anti-inflammatoryagent. Additional examples of drugs to be administered concurrentlyinclude but are not limited to antivirals, antibiotics, analgesics,corticosteroids, antagonists of inflammatory cytokines, non-steroidalanti-inflammatories, pentoxifylline, thalidomide, and disease-modifyingantirheumatic drugs (DMARDs) such as azathioprine, cyclophosphamide,cyclosporine, hydroxychloroquine sulfate, methotrexate, leflunomide,minocycline, penicillamine, sulfasalazine and gold compounds such asoral gold, gold sodium thiomalate, and aurothioglucose. Additionally, aCtLL polypeptide, a solCtLL polypeptide, fragment, antibody, or CtLLbinding partner may be combined with a second CtLL polypeptide, solCtLLpolypeptide, antibody against a CtLL polypeptide, or a CtLLpolypeptide-derived peptide that acts as a competitive inhibitor of anative a CtLL polypeptide.

Any efficacious route of administration may be used to therapeuticallyadminister a CtLL polypeptide, a solCtLL polypeptide, fragment,antibody, or CtLL binding partner thereof, including those compositionscomprising CtLL polynucleotides. Parenteral administration includesinjection, for example, via intra-articular, intravenous, intramuscular,intralesional, intraperitoneal or subcutaneous routes by bolus injectionor by continuous infusion. Other routes include localizedadministration, e.g., at a site of disease or injury. Other suitablemeans of administration include sustained release from implants; aerosolinhalation and/or insufflation; eyedrops; vaginal or rectalsuppositories; buccal preparations; oral preparations, including pills,syrups, lozenges or chewing gum; and topical preparations such aslotions, gels, sprays, ointments or other suitable techniques.Alternatively, a CtLL polypeptide, a solCtLL polypeptide, fragment,antibody, or CtLL binding partner may be delivered by implanting cellsthat express the polypeptide, for example, by implanting cells thatexpress a CtLL polypeptide, a solCtLL polypeptide, fragment, antibody,or CtLL binding partner. Cells may also be cultured ex vivo in thepresence of polypeptides of the invention in order to proliferate or toproduce a desired effect on or activity in such cells. Treated cells canthen be introduced in vivo for therapeutic purposes. In anotherembodiment, the subject's own cells are induced to produce a CtLLpolypeptide, a solCtLL polypeptide, fragment, antibody, or CtLL bindingpartner by transfection in vivo or ex vivo with a polynucleotide thatencodes a CtLL polypeptide, a solCtLL polypeptide, fragment, antibody,or CtLL binding partner. The polynucleotide can be introduced into thesubject's cells, for example, by injecting naked DNA orliposome-encapsulated DNA that encodes a CtLL polypeptide, a solCtLLpolypeptide, fragments thereof, antibody, or CtLL binding partner, or byother means of transfection. Polynucleotides of the invention may alsobe administered to subjects by other known methods for introduction ofnucleic acids into a cell or organism (including, without limitation, inthe form of viral vectors).

When a therapeutically effective amount of a CtLL polypeptide, solCtLLpolypeptide, fragments thereof, antibody, or binding partner of theinvention is administered orally, the polypeptide will typically be inthe form of a tablet, capsule, powder, solution or elixir. Whenadministered in tablet form, the pharmaceutical composition of theinvention may additionally contain a solid carrier such as a gelatin oran adjuvant. The tablet, capsule, and powder contain from about 5 to 95%a polypeptide of the invention, and preferably from about 25 to 90% apolypeptide of the invention. When administered in liquid form, a liquidcarrier such as water, petroleum, oils of animal or plant origin such aspeanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oilsmay be added. The liquid form of the pharmaceutical composition mayfurther contain physiological saline solution, dextrose or othersaccharide solution, or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol. When administered in liquid form, thepharmaceutical composition contains from about 0.5 to 90% by weight of apolypeptide of the invention, and preferably from about 1 to 50% apolypeptide of the invention.

When a therapeutically effective amount of a CtLL polypeptide, solCtLLpolypeptide, fragments thereof, antibody, or binding agent of theinvention is administered by intravenous, cutaneous or subcutaneousinjection, the polypeptide will be in the form of a pyrogen-free,parenterally acceptable aqueous solution. The preparation of suchparenterally acceptable polypeptide solutions, having due regard to pH,isotonicity, stability, and the like, is within the skill in the art. Apreferred pharmaceutical composition for intravenous, cutaneous, orsubcutaneous injection should contain, in addition to a polypeptide ofthe invention, an isotonic vehicle such as Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, Lactated Ringer's Injection, or other vehicle as known in theart. The pharmaceutical composition of the invention may also containstabilizers, preservatives, buffers, antioxidants, or other additivesknown to those of skill in the art. The duration of intravenous therapyusing the pharmaceutical composition of the invention will vary,depending on the severity of the disease being treated and the conditionand potential idiosyncratic response of each individual subject. It iscontemplated that the duration of each application of a polypeptide ofthe invention will be in the range of 12 to 24 hours of continuousintravenous administration. Ultimately the attending physician willdecide on the appropriate duration of intravenous therapy.

For compositions of the invention which are useful for tissue repair orregeneration, the therapeutic method includes administering apyrogen-free, physiologically acceptable form of the compositiontopically, systematically, locally or in association with an implant ordevice. Further, the composition may desirably be encapsulated orinjected in a viscous form for delivery to the site tissue damage.Additional useful agents may also optionally be included in thecomposition, as described above, or may be administered simultaneouslyor sequentially with the composition in the methods of the invention.The compositions can include a matrix capable of delivering thepolypeptide-containing composition to the site tissue damage, providinga structure for the developing tissue and optimally capable of beingresorbed into the body. The choice of matrix material is based onbiocompatibility, biodegradability, mechanical properties, cosmeticappearance and interface properties. Potential matrices for thecompositions include calcium sulfate, tricalciumphosphate,hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.Other potential matrices are nonbiodegradable and chemically defined,such as sintered hydroxyapatite, bioglass, aluminates, or otherceramics. Matrices may be comprised of combinations of any of the abovementioned types of material, such as polylactic acid and hydroxyapatiteor collagen and tricalciumphosphate. Progress can be monitored byperiodic assessment of tissue/bone growth and/or repair, for example,X-rays, histomorphometric determinations and tetracycline labeling.

In addition to human subjects, compositions comprising a CtLLpolypeptide, solCtLL polypeptide, fragments thereof, antibody, or CtLLbinding partner is useful in the treatment of disease conditions innon-human animals, such as pets (dogs, cats, birds, primates, and thelike), domestic farm animals (horses cattle, sheep, pigs, birds, and thelike). In such instances, an appropriate dose may be determinedaccording to the animal's body weight. For example, a dose of 0.2-1mg/kg may be used. Alternatively, the dose is determined according tothe animal's surface area, an exemplary dose ranging from 0.1-20 mg/m²,or more preferably, from 5-12 mg/m². For small animals, such as dogs orcats, a suitable dose is 0.4 mg/kg. In a one embodiment, a CtLLpolypeptide, solCtLL polypeptide, fragments thereof, antibody, or CtLLbinding partner (preferably constructed from genes derived from the samespecies as the subject), is administered by injection or other suitableroute one or more times per week until the animal's condition isimproved, or it may be administered indefinitely.

The invention also relates to the use of a CtLL polypeptide, a solCtLLpolypeptide, fragments thereof, and variants; polynucleotide encoding aCtLL polypeptide, a solCtLL polypeptide, fragments thereof, andvariants; agonists or antagonists of a CtLL polypeptide such asantibodies or solCtLL polypeptides; a CtLL polypeptide binding partner;complexes formed from a CtLL polypeptide, a solCtLL polypeptide,fragments thereof, variant, and binding partner, and the like, in themanufacture of a medicament for the prevention or therapeutic treatmentof a disease or disorder.

Further encompassed by the invention are systems and methods foranalyzing CtLL polypeptides comprising identifying and/or characterizingone or more CtLL polypeptides, polynucleotides encoding CtLLpolypeptides, and corresponding genes, these systems and methodspreferably comprising a data set representing a set of one or more CtLLmolecules. Accordingly, the invention provides a computer readablemedium having stored thereon a member selected from the group consistingof a polynucleotide comprising a sequence as set forth in SEQ ID NO:1,3, 5, 7, 9, 11, 13, and/or 15; or a polypeptide comprising a sequence asset forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, and/or 16.

One embodiment of the invention comprises a computing environment and aplurality of algorithms selectively executed to analyze a polypeptide orpolynucleotide of the invention. Examples of analyses of an CtLLpolypeptide include, without limitation, displaying the amino acidsequence of a polypeptide in the set, comparing the amino acid sequenceof one polypeptide in the set to the amino acid sequence of anotherpolypeptide in the set, predicting the structure of a polypeptide in theset, determining the nucleotide sequences of nucleic acids encoding apolypeptide in the set, and identifying a gene corresponding to apolypeptide in the set.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All headings and subheadingprovided herein are solely for ease of reading and should not beconstrued to limit the invention. The terms “a”, “an” and “the” as usedherein are meant to encompass the plural unless the context clearlydictates the singular form. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the invention, suitable methods and materials are describedbelow. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Thefollowing examples are intended to illustrate particular embodiments andnot to limit the scope of the invention.

EXAMPLES Example 1 Identification of CtLL, a New Member of the C-TypeLectin Family of Polypeptides

A data set was received from Celera Genomics (Rockville, Md.) containingamino acid sequences predicted to be encoded by the human genome. Thisdata set was threaded through a folding algorithm based upon the work ofAdam Godzik and Jeff Skolnick's “GeneFold” (distributed by Tripos)protein to identify helical cytokine family polypeptides. An amino acidsequence as set forth in SEQ ID NO:2 from amino acid 22 to 116 wasidentified in the search. A subsequent BLAST search using the foregoingsequence revealed a mouse cDNA (GenBank accession no. AK16908, laterrenamed as XP_(—)132881, which is incorporated herein by reference)predicted to encode a 275 amino acid protein. The BLAST search alsoidentified C-type lectin molecules. Alternative murine splice variantsof the minus exon 4 type were also identified which would generatemolecules without the C lectin domain but with new transmembranespanning regions.

By PCR a human C-type lectin molecule and additional splice variantswere identified, and these are presented in FIG. 1. The amino acidsequences presented in FIG. 1 are presented in standard 1-letter aminoacid code, where “A” represents alanine, “C” represents cysteine, andthe like.

Example 2 CtLL Polypeptide and Polynucleotide Analysis

Analysis of the polypeptide sequences of SEQ ID Nos:2, 4, 6, 8, 10, 12,14, and 16 indicated classic membrane anchor(s) and a type-2 topology.An alignment and computer algorithm analysis of SEQ ID Nos:2, 4, 6, 8,10, 12, 14, and 16 indicates a transmembrane domain includes about aminoacids 42 to 65 of SEQ ID Nos:2, 4, 6, 8, 10, 12, 14, and 16; includesabout amino acids 137 to 159 of SEQ ID Nos:2, 8, 14, and 16; andincludes about amino acids 164 to 182 of SEQ ID NO:2. In addition,soluble CtLL polypeptides (“solCtLL”) of the present invention compriseamino acids: from about residue 1 to about 41 of SEQ ID NO:2, 4, 6, 8,10, 12, 14, or 16; from about residue 66 to about 136 of SEQ ID NO:2, 8,14, and 16; from about residue x₁ to about x₂ of SEQ ID NO:4, wherein x₁is a residue between and including residues 66 and 143 and x₂ is aresidue between and including residue 226 and 233 (e.g., 226, 227, 228,229, etc.); from about residue x₁ to about x₂ of SEQ ID NO:6, wherein x₁is a residue between and including residues 66 and 143 and x₂ is aresidue between and including residue 227 and 232 (e.g., 227, 228, 229,230, etc.); from about residue x₁ to about 247 of SEQ ID NO:10, whereinx₁ is a residue between and including about residues 66 and 142; and/orfrom about residue x₁ to about 276 of SEQ ID NO:12, wherein x₁ is aresidue between and including residues 66 and 142. A putative lectindomain was identified in the polypeptides of the invention as comprisingan amino acid sequence as set forth from about 143 to 226 of SEQ IDNO:4; from about 143 to 227 of SEQ ID NO:6; from about 142 to 247 of SEQID NO:10; and from about 142 to 276 of SEQ ID NO:12. Furthermore anumber of putative glycosylation sites were identified and include N91and N101 of SEQ ID Nos:2, 4, 6, 8, 10, 12, 14, and 16.

The polynucleotide sequences encoding the CtLL polypeptides of SEQ IDNos:2, 4, 6, 8, 10, 12, 14, and 16 are provided. An analysis of thecoding sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, or 15, demonstratesthat the domain having ligand binding activity correspond, for example,to a sequence from about nucleotide 427 to 678 of SEQ ID NO:3, fromabout 463 to 717 of SEQ ID NO:5, from about 524 to 841 of SEQ ID NO:9,and from about 475 to 876 of SEQ ID NO:11. Accordingly, a polynucleotidecomprising the foregoing sequence above represent a coding sequence fora soluble CtLL polypeptide having oxidized lipid and/or carbohydratebinding activity.

Variants of the CtLL polypeptide sequences can be identified based uponthe sequences provided herein. Amino acid substitutions and otheralterations (deletions, insertions, and the like) to CtLL amino acidsequences are predicted to be more likely to alter or disrupt CtLLpolypeptide activities if they result in changes to the conservedresidues of the amino acid sequences as shown in FIG. 1, andparticularly if those changes do not substitute an amino acid of similarstructure (such as substitution of any one of the aliphaticresidues—Ala, Gly, Leu, Ile, or Val—for another aliphatic residue).Conversely, if a change is made to a CtLL amino acid sequence resultingin substitution of the residue at that position in the alignment fromone of the other CtLL polypeptide sequences, it is less likely that suchan alteration will affect the function of the altered CtLL polypeptide.

Example 3 Monoclonal Antibodies that Bind Polypeptides of the Invention

A substantially purified CtLL polypeptide or solCtLL polypeptide can beused to generate monoclonal antibodies immunoreactive therewith, usingconventional techniques such as those described in U.S. Pat. No.4,411,993. Mice are immunized with a CtLL polypeptide immunogenemulsified in complete Freund's adjuvant, and injected in amountsranging from 10-100 μg subcutaneously or intraperitoneally. Ten totwelve days later, the immunized animals are boosted with additionalCtLL polypeptide emulsified in incomplete Freund's adjuvant. Mice areperiodically boosted thereafter on a weekly to bi-weekly immunizationschedule. Serum samples are periodically taken by retro-orbital bleedingor tail-tip excision to test for a CtLL polypeptide antibody by dot blotassay, ELISA (Enzyme-Linked Immunosorbent Assay) or inhibition ofbinding of a CtLL polypeptide to a CtLL polypeptide binding partner.

Following detection of an appropriate antibody titer, positive animalsare provided one last intravenous injection of a CtLL polypeptide insaline. Three to four days later, the animals are sacrificed, spleencells harvested, and spleen cells are fused to a murine myeloma cellline, e.g., NS1 or preferably P3x63Ag8.653 (ATCC CRL 1580). Fusionsgenerate hybridoma cells, which are plated in multiple microtiter platesin a HAT (hypoxanthine, aminopterin and thymidine) selective medium toinhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

The hybridoma cells are screened by ELISA for reactivity against asubstantially pure CtLL polypeptide by adaptations of the techniquesdisclosed in Engvall et al., (Immunochem. 8:871, 1971) and in U.S. Pat.No. 4,703,004. A preferred screening technique is the antibody capturetechnique described in Beckmann et al., (J. Immunol. 144:4212, 1990).Positive hybridoma cells can be injected intraperitoneally intosyngeneic BALB/c mice to produce ascites containing high concentrationsof anti-CtLL monoclonal antibody. Alternatively, hybridoma cells can begrown in vitro in flasks or roller bottles by various techniques.Monoclonal antibodies produced in mouse ascites can be purified byammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to Polypeptide A or Polypeptide G can also be used,as can chromatography based upon binding to CtLL polypeptide.

Example 4 Chromosome Mapping

The gene corresponding to a CtLL polypeptide is mapped using PCR-basedmapping strategies. Initial human chromosomal assignments are made usingCtLL-specific PCR primers and a BIOS Somatic Cell Hybrid PCRable DNA kitfrom BIOS Laboratories (New Haven, Conn.), following the manufacturer'sinstructions. More detailed mapping is performed using a Genebridge 4Radiation Hybrid Panel (Research Genetics, Huntsville, Ala. (see, e.g.,Walter, M A et al., Nature Genetics 7:22-28, 1994). Data from thisanalysis is then submitted electronically to the MIT Radiation HybridMapper (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl)following the instructions contained therein. This analysis yieldsspecific genetic marker names which, when submitted electronically toNCBI: ncbi.nlm.nih.gov/genemap/map.cgi?CHR=19) yield the specificchromosome interval. The chromosomal location of the CtLL is onchromosome 19. The CtLL sequences lie closest in the genome to CLEC2 (afamily member expressed in liver) and between CLEC2 and CLEC1 (a familymember expressed in dendritic cells and endothelial cells in asub-cluster of molecules expressed in macrophages, DCs and endothelialcells including OLR1, which is expressed in macrophages and induced inendothelial cells and vascular smooth muscle cells. The order beginningfrom the end of the short arm of chromosome 19 and transcriptionorientation (→←) as well as ITIM presence are as follows:

Macrophage antigen←Macrophage Ag-2(AAW40215)(ITIM)CLEC2(ITIM)→←CtLL(ITIM)CLEC1→dectin1(ITAM)→OLR1→

Example 5 Hybridization Probes

Hybridization probes derived from SEQ ID NO:1, 3, 5, 7, 9, 11, 13, or 15are employed to screen cDNAs, genomic DNAs, or mRNAs. Although thelabeling of oligonucleotides, consisting of about 20 base-pairs, isspecifically described, essentially the same procedure is used withlarger cDNA fragments. Oligonucleotides are designed using OLIGO,labeled by combining 50 pmol of each oligomer and 250 mCi ofγ-³²P-adenosine triphosphate (Amersham, Chicago Ill.) and T4polynucleotide kinase (DuPont NEN®, Boston Mass.). The labeledoligonucleotides are substantially purified with Sephadex G-25 superfine resin column (Pharmacia). A portion containing 10⁷ counts perminute of each of the sense and antisense oligonucleotides is used in atypical membrane based hybridization analysis of human genomic DNAdigested with one of the following endonucleases (Ase I, Bgl II, Eco RI,Pst I, Xba 1, or Pvu II; DuPont NEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1.×SSC and0.5% SDS. Radiographic film is exposed to the blots and hybridizationpatterns are compared visually.

Example 6 Expression of CtLL Ligand Binding Domain

Expression of the solCtLL ligand binding domain is accomplished bysubcloning a corresponding polynucleotide coding sequence (e.g., asequence from about nucleotide 427 to 678 of SEQ ID NO:3, from about 463to 717 of SEQ ID NO:5, from about 524 to 841 of SEQ ID NO:9, or fromabout 475 to 876 of SEQ ID NO:11) into appropriate expression vector andtransfecting the vectors into suitable host cells.

Lectin activity of CtLL or biologically active fragments thereof may beassayed by first labeling the CtLL protein or polypeptide with ¹²⁵IBolton-Hunter reagent (Bolton and Hunter, Biochem J 133:529, 1973).Candidate ligands (including polysaccharides, glycoproteins or wholecells) previously arrayed in the wells of a 96 well plate are incubatedwith the labeled CtLL, washed and any wells with labeled CtLL complexare assayed. Data obtained using different concentrations of CtLL areused to calculate values for the number, affinity, and association ofCtLL with the candidate ligands.

Example 7 Analysis of Murine CtLL Polypeptide Expression by Real-TimeQuantitative PCR

Expression of the full length murine analog of full length CtLL(sequence available in Genebank Accession number XP_(—)132881) wasanalyzed in normal adult mice as well as in an asthma mouse model.

For the following experiments, RNA samples were obtained from a varietyof murine tissues, or from cells or tissues treated with a variety ofcompounds. The RNA samples were DNase treated (product #1906, Ambion,Austin, Tex.), and reverse transcribed into a population of cDNAmolecules using TaqMan Reverse Transcription Reagents (product#N808-0234, Applied Biosystems, Foster City, Calif.) according to themanufacturer's instructions using random hexamers. Each population ofcDNA molecules was placed into specific wells of a multi-well plate ateither 5 ng or 20 ng per well and run in triplicate. Pooling was usedwhen same tissue types and stimulation conditions were applied butcollected from different donors. Negative control wells were included ineach multi-well plate of samples.

Sets of probes and oligonucleotide primers complementary to mRNAsencoding the murine analog of full length CtLL polypeptide were designedusing Primer Express software (Applied Biosystems, Foster City, Calif.)and synthesized, and PCR conditions for these probe/primer sets wereoptimized to produce a steady and logarithmic increase in PCR productevery thermal cycle between approximately cycle 20 and cycle 36. Theforward murine CtLL polypeptide primer used was 5′ GGG AAT CGG GAT GGAAAT AAC 3′ (SEQ ID NO:29); the reverse murine CtLL primer used was 5′GCG CCT CGC CAA AGT G 3′ (SEQ ID NO:30); and the labeled probe used forCtLL polypeptide was 5′ AAG AAG GAC ACC CAG CTC 3′ (SEQ ID NO:31).Oligonucleotide primers complementary to 18S RNA and to mRNAs encodingthe ‘housekeeper’ proteins, in this instance, muHPRT (hypoxanthinephosphoribosyltransferase), were synthesized and PCR conditions wereoptimized for these primer sets also. Multiplex TaqMan PCR reactionsusing both CtLL polypeptide and muHPRT probe/primer sets were set up in25-microliter volumes with TaqMan Universal PCR Master Mix (product#4304437, Applied Biosystems, Foster City, Calif.) on an AppliedBiosystems Prism 7700 Sequence Detection System. Threshold cycle values(C_(T)) were determined using Sequence Detector software version 1.7a(Applied Biosystems, Foster City, Calif.), and delta C_(T) wascalculated and transformed to 2e(−dC_(T)), which is 2 to the minus deltaC_(T), for relative expression comparison of CtLL to muHPRT. Forexample, a ratio of expression of 0.0136 indicates that the expressionof CtLL is 1.36% that of the housekeeping gene.

In one set of experiments, expression of murine CtLL polypeptiderelative to muHPRT was analyzed in a variety of normal adult mousetissue samples. RNA was extracted from various tissues of adult C57B1/6mice (in all cases samples were pools from multiple mice) and expressionof CtLL compared with that of muHPRT. This analysis indicated that CtLLmessages are detectable and express at less than 10% of housekeepermRNAs in many adult tissues, including thymus, spleen, bone marrow,brain, spinal chord, kidney, skeletal muscle, heart, uterus, andovaries. The highest expression levels was found in the lymph nodes at12% of housekeeper; the lung, at 78% of housekeeper, and in particular,the testis, at 834% of housekeeping.

CtLL polypeptide expression relative to houekeeper gene expression intissue samples from a mouse asthma model was also analyzed. Experimentsusing C57B1/6 mice were performed as follows. Naïve mice received nopriming and no challenge. Unchallenged and challenged mice were primedwith an intranasal exposure to 10 ug ovalbumin at day −21 and −14prechallenge. Challenged mice were exposed intranasally to ovalbumin at100 ug/50 ul on a one dose, two dose, or three dose schedule over aperiod of up to 48 hours; then sacrificed at 96 hr, the lungs removed,and expression of murine CtLL polypeptide analyzed as described above.The results of this analysis are presented in Table 2 below. The CB andDS designation refers to two separate sets of experiments. It wasgenerally found that expression of murine CttLL was highest in naïve andunchallenged mice, and that expression was down-regulated by 3-4 timesin a challenged animal compared with the expression in naïve orunchalleged animals.

TABLE 2 muCtLL muHPRT Sample Avg CT Avg CT 2e-dCT Minus Err Plus ErrCB1102 48 h dose3 A1 33.4633 29.4167 0.0605107 0.0520026 0.0704108CB1102 48 h dose3 A2 32.7133 29.5267 0.1098292 0.0971053 0.1242202CB1102 24 h dose3 B1 32.35 28.73 0.0813339 0.0761669 0.0868514 CB1102 24h dose3 B2 31.76 28.39 0.0967228 0.0789843 0.1184451 CB1102 24 h dose1C1 31 28.4733 0.1735392 0.1601695 0.1880249 CB1102 24 h dose1 C2 31.9528.7533 0.1090705 0.0969641 0.1226885 CB1102 4 h dose1 D1 30.5533 27.840.1524773 0.1367968 0.1699553 CB1102 4 h dose1 D2 30.5033 28.26330.2116863 0.2044913 0.2191345 CB1102 no chal E1 30.24 28.21 0.24485510.2365693 0.2534311 CB1102 no chal E2 30.2533 27.75 0.1763687 0.15820790.1966142 CB1102 Naive F1 30.67 28.4 0.2073299 0.2029856 0.2117671CB1102 Naive F2 30.0433 27.67 0.1929992 0.1750592 0.2127777 DS0602 48 hdose3 A1 32.2067 28.8233 0.095833 0.084875 0.1082058 DS0602 48 h dose3A2 30.8933 28.21 0.1556812 0.14785 0.1639273 DS0602 24 h dose3 B1 31.6128.1767 0.0925686 0.089209 0.0960547 DS0602 24 h dose3 B2 30.646727.1067 0.0859714 0.0796628 0.0927795 DS0602 24 h dose1 C1 29.97 28.060.2660926 0.2394133 0.2957448 DS0602 24 h dose1 C2 29.9033 27.93330.255253 0.2418856 0.2693592 DS0602 4 h dose1 D1 30.9533 29.56670.3824474 0.3586521 0.4078215 DS0602 4 h dose1 D2 29.8567 27.970.2704312 0.2645359 0.2764578 DS0602 no chal E1 29.77 28.1467 0.32458470.2875334 0.3664102 DS0602 no chal E2 30.13 28.1367 0.2511579 0.24095780.2617899 DS0602 Naive F1 29.08 27.8333 0.4214208 0.4090606 0.4341545DS0602 Naive F2 29.64 28.3433 0.4070656 0.3827274 0.4329516 Negative 4040 0 0 0

These results may suggest that during inflammation, macrophageprocessing of apoptotic cells is downregulated, perhaps to protectagainst excessive autoantigen presentation. This would be consistantwith the lower level of CtLL expression when macrophages are activatedin vitro.

Example 8 Analysis of CtLL Polypeptide Expression in Human Samples byReal-Time Quantitative PCR

RNA samples from human tissues were obtained commercially (Ambion,Austin, Tex.). The RNA samples were DNase treated (product #1906,Ambion, Austin, Tex.), and reverse transcribed into a population of cDNAmolecules using TaqMan Reverse Transcription Reagents (product#N808-0234, Applied Biosystems, Foster City, Calif.) according to themanufacturer's instructions using random hexamers as described above.Each population of cDNA molecules was placed into specific wells of amulti-well plate at either 5 ng or 20 ng per well and run in triplicate.Pooling was used when same tissue types and stimulation conditions wereapplied but collected from different donors. Negative control wells wereincluded in each multi-well plate of samples.

Sets of probes and oligonucleotide primers complementary to mRNAsencoding a CtLL polypeptide (SEQ ID NO: 12) were designed using PrimerExpress software (Applied Biosystems, Foster City, Calif.) andsynthesized, and PCR conditions for these probe/primer sets wereoptimized to produce a steady and logarithmic increase in PCR productevery thermal cycle between approximately cycle 20 and cycle 36. Theforward CtLL primer used was 5′ AGT GTA CTG AAG AGG CAG GAA CAA 3′ (SEQID NO:32); the reverse CtLL primer used was 5′ GAC ATG GAT TAC ATC TGTGGT CTG A 3′ (SEQ ID NO:33); and the labeled probe used for CtLLpolypeptide was 5′ TGG CCA TCA AAC TGT GCC AAG AGC 3′ (SEQ ID NO:34).Oligonucleotide primers complementary to 18S RNA and to mRNAs encodingeither HPRT (hypoxanthine phosphoribosyltransferase) or β-actin weresynthesized and PCR conditions were optimized for these primer setsalso. Multiplex TaqMan PCR reactions using both human CtLL polypeptideand probe/primer sets were set up in 25-microliter volumes with TaqManUniversal PCR Master Mix (product #4304437, Applied Biosystems, FosterCity, Calif.) on an Applied Biosystems Prism 7700 Sequence DetectionSystem. Threshold cycle values (C_(T)) were determined using SequenceDetector software version 1.7a (Applied Biosystems, Foster City,Calif.), and delta C_(T) was calculated and transformed to 2e(−dC_(T)),which is 2 to the minus delta C_(T), for relative expression comparisonof CtLL to HPRT or β-actin.

Analysis of CtLL expression relative to HPRT was performed using normalhuman tissue samples. The following results were obtained. Expressionwas found at 28.38% of housekeeper for testis, 2.76% for spleen, 116% ofhousekeeping for skin, 6.58% for lung, and 1.56% for spinal chord. Theseresults are presented in Table 3.

TABLE 3 huCtLL HPRT Sample Avg CT Avg CT 2e-dCT Minus Err Plus Err SmIntestine 37.07 29.48 0 0 0 Stomach 40 30.28 0 0 0 Testis 28.17 26.3530.2838761 0.2626444 0.3068242 Colon 38.6467 30.05 0 0 0 Prostate 4029.803 0 0 0 Adrenal 40 29.617 0 0 0 Ovary 40 29.273 0 0 0 Pancreas39.68 30.3 0 0 0 Sk Muscle 40 31.703 0 0 0 Spleen 40 31.65 0 0 0 Thymus37.3333 30.443 0 0 0 F Stomach 39.0667 31.067 0 0 0 F Spleen 34.846729.67 0.0276483 0.0217914 0.0350793 F Lung 39.8567 32.62 0 0 0 F Colon37.76 30.153 0 0 0 Thyroid 36.6333 30.263 0 0 0 Skin 31.0367 31.2531.1620456 1.1172113 1.2086791 F Kidney 39.58 29.697 0 0 0 F Sk Muscle 4030.5 0 0 0 Uterus 37.3767 30.29 0 0 0 Brain 39.7 29.873 0 0 0 Liver 4034.263 0 0 0 Heart 40 34.623 0 0 0 Kidney 40 30.073 0 0 0 Lung 35.831.873 0.0657591 0.0522674 0.0827332 Trachea 38.3333 30.59 0 0 0Cerebellum 40 29.19 0 0 0 F Brain 40 28.593 0 0 0 Spinal Cord 35.6129.603 0.015553 0.0137479 0.0175951 F Liver 39.2367 29.303 0 0 0Placenta 39.7133 33.193 0 0 0 NEG 40 40 0 0 0

In other expression analyses, no expression was detected in dendriticcells. However, a low but significant CtLL expression (0.001079% ofβ-Actin) was detected in monocytes. No detectable expression was foundin bone tissue or adipose tissue, or in various tissues subjected totreatments using a variety of cytokines and other factors.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A substantially purified monoclonal antibody that specifically bindsto the polypeptide consisting of the lectin domain amino acid sequencefrom residue 142 to residue 276 of SEQ ID NO:12.
 2. The antibody ofclaim 1, wherein the antibody is a human or humanized antibody.